AAV Vectors Targeted to Oligodendrocytes

ABSTRACT

The invention relates to chimeric AAV capsids targeted to oligodendrocytes, virus vectors comprising the same, and methods of using the vectors to target oligodendrocytes.

STATEMENT OF PRIORITY

This application claims the benefit of U.S. Provisional Application Ser.No. 61/707,108, filed Sep. 28, 2012, the entire contents of which isincorporated by reference herein.

FIELD OF THE INVENTION

The invention relates to chimeric AAV capsids targeted tooligodendrocytes, virus vectors comprising the same, and methods ofusing the vectors to target oligodendrocytes.

BACKGROUND OF THE INVENTION

Adeno-associated virus (AAV) was first reported to efficiently transducemuscle over ten years ago (Xiao et al., (1996) J. Virol. 70:8098-8108).The recombinant AAV (rAAV) genome composed of a foreign expressioncassette and AAV inverted terminal repeat (ITR) sequences exists ineukaryotic cells in an episomal form that is responsible for persistenttransgene expression (Schnepp et al., (2003) J. Virol. 77:3495-3504).AAV vectors have a good safety profile. No human disease has beenassociated with wild-type AAV infection and low toxicity is observed inhuman subjects following transduction by rAAV (Manno et al., (2003)Blood 101:2963-2972).

In the brain, the vast majority of AAV vectors exhibit a dominantpreference for neurons with a very low efficacy for other cell types,such as oligodendrocytes. Recent advances in AAV engineering anddirected evolution have expanded the ability to develop novel AAVserotypes, including vectors with altered tropism (Gray et al., (2010)Mol. Ther. 18:570-578). However, in the central nervous system, all AAVvectors and chimeras, except AAV4, exhibit a dominant neuronal tropism.AAV vectors that efficiently target oligodendrocytes have not beendeveloped.

SUMMARY OF THE INVENTION

The present invention is based, in part, on the development of achimeric AAV capsid sequence that exhibits a dominant tropism foroligodendrocytes. The chimeric capsid can be used to create AAV vectorsthat transduce oligodendrocytes in the central nervous system (CNS) ofsubjects.

Thus, one aspect of the invention relates to a nucleic acid encoding anAAV capsid, the nucleic acid comprising an AAV capsid coding sequencethat is at least 90% identical to: (a) the nucleotide sequence of SEQ IDNO:1; or (b) a nucleotide sequence encoding any one of SEQ ID NOS:2-4,along with cells and viral particles comprising the nucleic acid.

Another aspect of the invention relates to an AAV capsid comprising anamino acid sequence at least 96% identical to any one of SEQ ID NOS:2-4,along with AAV particles comprising an AAV vector genome and the AAVcapsid of the invention.

A further aspect of the invention relates to a method of producing arecombinant AAV particle comprising an AAV capsid, the methodcomprising: providing a cell in vitro with a nucleic acid of theinvention, an AAV rep coding sequence, an AAV vector genome comprising aheterologous nucleic acid, and helper functions for generating aproductive AAV infection; and allowing assembly of the recombinant AAVparticle comprising the AAV capsid and encapsidating the AAV vectorgenome.

An additional aspect of the invention relates to a pharmaceuticalformulation comprising the nucleic acid, virus particle, AAV capsid, orAAV particle of the invention in a pharmaceutically acceptable carrier.

Another aspect of the invention relates to a method of delivering anucleic acid of interest to an oligodendrocyte, the method comprisingcontacting the oligodendrocyte with the AAV particle of the invention.

A further aspect of the invention relates to a method of delivering anucleic acid of interest to an oligodendrocyte in a mammalian subject,the method comprising administering an effective amount of the AAVparticle or pharmaceutical formulation of the invention to a mammaliansubject.

An additional aspect of the invention relates to a method of deliveringa nucleic acid of interest to an area of the CNS bordering a compromisedblood brain barrier area in a mammalian subject, the method comprisingintravenously administering an effective amount of the AAV particle orpharmaceutical formulation of the invention to a mammalian subject.

Another aspect of the invention relates to a method of treating adisorder associated with oligodendrocyte dysfunction in a mammaliansubject in need thereof, the method comprising administering atherapeutically effective amount of the AAV particle or pharmaceuticalformulation of the invention to a mammalian subject.

A further aspect of the invention relates to a method of preparing anAAV capsid having a tropism profile of interest, the method comprisingmodifying the AAV capsid of the invention to insert an amino acidsequence providing the tropism profile of interest.

These and other aspects of the invention are set forth in more detail inthe description of the invention below.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the chimeric structure of the BNP61, BNP62, and BNP63 AAVcapsid clones.

FIGS. 2A-2C show the tropism of BNP61 for oligodendrocytes in ratcaudate. (A) shows GFP positive oligodendrocytes in the rat caudate 1week after the infusion of BNP61-CBh-GFP vectors. Note that there are noGFP positive neurons. (B) shows a higher magnification that reflectsclear oligodendrocyte morphology, and (C) shows that none of the GFPpositive cells colocalize with the cellular marker for astrocytes,GFAP(red).

FIGS. 3A-3B show the tropism of BNP61 for oligodendrocytes in primaryoligodendrocyte cultures.

FIG. 4 shows the biodistribution of BNP61 (MG001) and parent capsidsafter intravenous injection in female wild-type mice, determined byquantitative PCR. The Y-axis is copies of GFP per diploid mouse genome.

FIG. 5 shows that BNP61 crosses the compromised blood-brain barrierafter peripheral administration.

FIG. 6 shows the tropism of BNP63 for oligodendrocytes in rat piriformcortex.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is based, in part, on the development of achimeric AAV capsid sequence that exhibits a tropism foroligodendrocytes. The chimeric capsid can be used to create AAV vectorsthat transduce oligodendrocytes in the CNS of subjects.

The present invention is explained in greater detail below. Thisdescription is not intended to be a detailed catalog of all thedifferent ways in which the invention may be implemented, or all thefeatures that may be added to the instant invention. For example,features illustrated with respect to one embodiment may be incorporatedinto other embodiments, and features illustrated with respect to aparticular embodiment may be deleted from that embodiment. In addition,numerous variations and additions to the various embodiments suggestedherein will be apparent to those skilled in the art in light of theinstant disclosure which do not depart from the instant invention.Hence, the following specification is intended to illustrate someparticular embodiments of the invention, and not to exhaustively specifyall permutations, combinations and variations thereof.

Unless the context indicates otherwise, it is specifically intended thatthe various features of the invention described herein can be used inany combination. Moreover, the present invention also contemplates thatin some embodiments of the invention, any feature or combination offeatures set forth herein can be excluded or omitted. To illustrate, ifthe specification states that a complex comprises components A, B and C,it is specifically intended that any of A, B or C, or a combinationthereof, can be omitted and disclaimed singularly or in any combination.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. The terminology used in thedescription of the invention herein is for the purpose of describingparticular embodiments only and is not intended to be limiting of theinvention.

Nucleotide sequences are presented herein by single strand only, in the5′ to 3′ direction, from left to right, unless specifically indicatedotherwise. Nucleotides and amino acids are represented herein in themanner recommended by the IUPAC-IUB Biochemical Nomenclature Commission,or (for amino acids) by either the one-letter code, or the three lettercode, both in accordance with 37 C.F.R. §1.822 and established usage.

Except as otherwise indicated, standard methods known to those skilledin the art may be used for production of recombinant and syntheticpolypeptides, antibodies or antigen-binding fragments thereof,manipulation of nucleic acid sequences, production of transformed cells,the construction of rAAV constructs, modified capsid proteins, packagingvectors expressing the AAV rep and/or cap sequences, and transiently andstably transfected packaging cells. Such techniques are known to thoseskilled in the art. See, e.g., SAMBROOK et al., MOLECULAR CLONING: ALABORATORY MANUAL 2nd Ed. (Cold Spring Harbor, N.Y., 1989); F. M.AUSUBEL et al. CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (Green PublishingAssociates, Inc. and John Wiley & Sons, Inc., New York).

All publications, patent applications, patents, nucleotide sequences,amino acid sequences and other references mentioned herein areincorporated by reference in their entirety.

1. Definitions

The designation of all amino acid positions in the AAV capsid subunitsin the description of the invention and the appended claims is withrespect to VP1 capsid subunit numbering.

As used in the description of the invention and the appended claims, thesingular forms “a,” “an” and “the” are intended to include the pluralforms as well, unless the context clearly indicates otherwise.

As used herein, “and/or” refers to and encompasses any and all possiblecombinations of one or more of the associated listed items, as well asthe lack of combinations when interpreted in the alternative (“or”).

Moreover, the present invention also contemplates that in someembodiments of the invention, any feature or combination of features setforth herein can be excluded or omitted.

Furthermore, the term “about,” as used herein when referring to ameasurable value such as an amount of a compound or agent of thisinvention, dose, time, temperature, and the like, is meant to encompassvariations of ±20%, ±10%, ±5%, ±1%, ±0.5%, or even ±0.1% of thespecified amount.

The term “consisting essentially of” as used herein in connection with anucleic acid, protein or capsid structure means that the nucleic acid,protein or capsid structure does not contain any element other than therecited element(s) that significantly alters (e.g., more than about 1%,5% or 10%) the function of interest of the nucleic acid, protein orcapsid structure, e.g., tropism profile of the protein or capsid or aprotein or capsid encoded by the nucleic acid.

The term “adeno-associated virus” (AAV) in the context of the presentinvention includes without limitation AAV type 1, AAV type 2, AAV type 3(including types 3A and 3B), AAV type 4, AAV type 5, AAV type 6, AAVtype 7, AAV type 8, AAV type 9, AAV type 10, AAV type 11, avian AAV,bovine AAV, canine AAV, equine AAV, and ovine AAV and any other AAV nowknown or later discovered. See, e.g., BERNARD N. FIELDS et al.,VIROLOGY, volume 2, chapter 69 (4th ed., Lippincott-Raven Publishers). Anumber of additional AAV serotypes and clades have been identified (see,e.g., Gao et al., (2004) J. Virol. 78:6381-6388 and Table 1), which arealso encompassed by the term “AAV.”

The genomic sequences of various AAV and autonomous parvoviruses, aswell as the sequences of the ITRs, Rep proteins, and capsid subunits areknown in the art. Such sequences may be found in the literature or inpublic databases such as the GenBank database. See, e.g., GenBank®Accession Numbers NC 002077, NC 001401, NC 001729, NC 001863, NC 001829,NC 001862, NC 000883, NC 001701, NC 001510, AF063497, U89790, AF043303,AF028705, AF028704, J02275, J01901, 102275, X01457, AF288061, AH009962,AY028226, AY028223, NC 001358, NC 001540, AF513851, AF513852, AY530579,AY631965, AY631966; the disclosures of which are incorporated herein intheir entirety. See also, e.g., Srivistava et al., (1983) J. Virol45:555; Chiorini et al., (1998) J. Virol. 71:6823; Chiorini et al.,(1999) J. Virol. 73:1309; Bantel-Schaal et al., (1999) J. Virol. 73:939;Xiao et al., (1999) J. Virol. 73:3994; Muramatsu et al., (1996) Virology221:208; Shade et at, (1986) J. Virol 58:921; Gao et al., (2002) Proc.Nat. Acad. Sci. USA 99:11854; international patent publications WO00/28061, WO 99/61601, WO 98/11244; U.S. Pat. No. 6,156,303; thedisclosures of which are incorporated herein in their entirety. See alsoTable 1. An early description of the AAV1, AAV2 and AAV3 terminal repeatsequences is provided by Xiao, X., (1996), “Characterization ofAdeno-associated virus (AAV) DNA replication and integration,” Ph.D.Dissertation, University of Pittsburgh, Pittsburgh, Pa. (incorporatedherein it its entirety).

TABLE 1 GenBank GenBank GenBank Accession Accession Accession CompleteGenomes Number Number Number Hu T88 AY695375 Hu42 AY530605Adeno-associated virus 1 NC_002077, AF063497 Hu T71 AY695374 Hu67AY530627 Adeno-associated virus 2 NC_001401 Hu T70 AY695373 Hu40AY530603 Adeno-associated virus 3 NC_001729 Hu T40 AY695372 Hu41AY530604 Adeno-associated virus NC_001863 Hu T32 AY695371 Hu37 AY5306003B Adeno-associated virus 4 NC_001829 Hu T17 AY695370 Rh40 AY530559Adeno-associated virus 5 Y18065, AF085716 Hu LG15 AY695377 Rh2 AY243007Adeno-associated virus 6 NC_001862 Clade C Bb1 AY243023 Avian AAV ATCCVR- AY186198, AY629583, Hu9 AY530629 Bb2 AY243022 865 NC_004828 AvianAAV strain DA-1 NC_006263, AY629583 Hu10 AY530576 Rh10 AY243015 BovineAAV NC_005889, AY388617 Hu11 AY530577 Hu17 AY530582 Clade A Hu53AY530615 Hu6 AY530621 AAV1 NC_002077, AF063497 Hu55 AY530617 Rh25AY530557 AAV6 NC_001862 Hu54 AY530616 Pi2 AY530554 Hu.48 AY530611 Hu7AY530628 Pi1 AY530553 Hu 43 AY530606 Hu18 AY530583 Pi3 AY530555 Hu 44AY530607 Hu15 AY530580 Rh57 AY530569 Hu 46 AY530609 Hu16 AY530581 Rh50AY530563 Clade B Hu25 AY530591 Rh49 AY530562 Hu. 19 AY530584 Hu60AY530622 Hu39 AY530601 Hu. 20 AY530586 Ch5 AY243021 Rh58 AY530570 Hu 23AY530589 Hu3 AY530595 Rh61 AY530572 Hu22 AY530588 Hu1 AY530575 Rh52AY530565 Hu24 AY530590 Hu4 AY530802 Rh53 AY530566 Hu21 AY530587 Hu2AY530585 Rh51 AY530564 Hu27 AY530592 Hu61 AY530623 Rh64 AY530574 Hu28AY530593 Clade D Rh43 AY530560 Hu 29 AY530594 Rh62 AY530573 AAV8AF513852 Hu63 AY530624 Rh48 AY530561 Rh8 AY242997 Hu64 AY530625 Rh54AY530567 Rh1 AY530556 Hu13 AY530578 Rh55 AY530568 Clade F Hu56 AY530618Cy2 AY243020 Hu14 (AAV9) AY530579 Hu57 AY530619 AAV7 AF513851 Hu31AY530596 Hu49 AY530612 Rh35 AY243000 Hu32 AY530597 Hu58 AY530620 Rh37AY242998 Clonal Isolate Hu34 AY530598 Rh36 AY242999 AAV6 Y18065,AF085716 Hu35 AY530599 Cy6 AY243016 AAV 3 NC_001729 AAV2 NC_001401 Cy4AY243018 AAV 3B NC_001863 Hu45 AY530608 Cy3 AY243019 AAV4 NC_001829 Hu47AY530610 Cy5 AY243017 Rh34 AY243001 Hu51 AY530613 Rh13 AY243013 Rh33AY243002 Hu52 AY530614 Clade E Rh32 AY243003 Hu T41 AY695378 Rh38AY530558 Hu S17 AY695376 Hu66 AY530626

A “chimeric” AAV nucleic acid capsid coding sequence or AAV capsidprotein is one that combines portions of two or more capsid sequences. A“chimeric” AAV virion or particle comprises a chimeric AAV capsidprotein.

The term “tropism” as used herein refers to preferential entry of thevirus into certain cell or tissue type(s) and/or preferentialinteraction with the cell surface that facilitates entry into certaincell or tissue types, optionally and preferably followed by expression(e.g., transcription and, optionally, translation) of sequences carriedby the viral genome in the cell, e.g., for a recombinant virus,expression of the heterologous nucleotide sequence(s). Those skilled inthe art will appreciate that transcription of a heterologous nucleicacid sequence from the viral genome may not be initiated in the absenceof trans-acting factors, e.g., for an inducible promoter or otherwiseregulated nucleic acid sequence. In the case of a rAAV genome, geneexpression from the viral genome may be from a stably integratedprovirus and/or from a non-integrated episome, as well as any other formwhich the virus nucleic acid may take within the cell.

The term “tropism profile” refers to the pattern of transduction of oneor more target cells, tissues and/or organs. Representative examples ofchimeric AAV capsids have a tropism profile characterized by efficienttransduction of oligodendrocytes with only low transduction of neurons,astrocytes, and other CNS cells.

The term “specific for oligodendrocytes” as used herein refers to aviral vector that, when administered directly into the CNS,preferentially transduces oligodendrocytes over neurons, astrocytes, andother CNS cell types. In some embodiments, at least about 80% of thetransduced cells are oligodendrocytes, e.g., at least about 85%, 90%,95%, 96%, 97%, 98%, 99% or more oligodendrocytes.

The term “disorder associated with oligodendrocyte dysfunction” as usedherein refers to a disease, disorder, or injury in whicholigodendrocytes are damaged, lost, or function improperly. The termincludes diseases, disorders, and injuries in which oligodendmcytes aredirectly affected as well as diseases, disorders, and injuries in whicholigodendrocytes become dysfunctional secondary to damage to other cells(e.g., spinal cord injury).

The term “bordering a compromised blood-brain barrier area” as usedherein refers to CNS cells that are adjacent to a portion of theblood-brain barrier in which the barrier function has been compromised.

As used herein, “transduction” of a cell by a virus vector (e.g., an AAVvector) means entry of the vector into the cell and transfer of geneticmaterial into the cell by the incorporation of nucleic acid into thevirus vector and subsequent transfer into the cell via the virus vector.

Unless indicated otherwise, “efficient transduction” or “efficienttropism,” or similar terms, can be determined by reference to a suitablepositive or negative control (e.g., at least about 50%, 60%, 70%, 80%,85%, 90%, 95% or more of the transduction or tropism, respectively, of apositive control or at least about 110%, 120%, 150%, 200%, 300%, 500%,1000% or more of the transduction or tropism, respectively, of anegative control).

Similarly, it can be determined if a virus “does not efficientlytransduce” or “does not have efficient tropism” for a target tissue, orsimilar terms, by reference to a suitable control. In particularembodiments, the virus vector does not efficiently transduce (i.e., doesnot have efficient tropism) for liver, kidney, gonads and/or germ cells.In particular embodiments, undesirable transduction of tissue(s) (e.g.,liver) is 20% or less, 10% or less, 5% or less, 1% or less, 0.1% or lessof the level of transduction of the desired target tissue(s) (e.g.,skeletal muscle, diaphragm muscle and/or cardiac muscle).

As used herein, the term “polypeptide” encompasses both peptides andproteins, unless indicated otherwise.

A “nucleic acid” or “nucleotide sequence” is a sequence of nucleotidebases, and may be RNA, DNA or DNA-RNA hybrid sequences (including bothnaturally occurring and non-naturally occurring nucleotide), but ispreferably either single or double stranded DNA sequences.

As used herein, an “isolated” nucleic acid or nucleotide sequence (e.g.,an “isolated DNA” or an “isolated RNA”) means a nucleic acid ornucleotide sequence separated or substantially free from at least someof the other components of the naturally occurring organism or virus,for example, the cell or viral structural components or otherpolypeptides or nucleic acids commonly found associated with the nucleicacid or nucleotide sequence.

Likewise, an “isolated” polypeptide means a polypeptide that isseparated or substantially free from at least some of the othercomponents of the naturally occurring organism or virus, for example,the cell or viral structural components or other polypeptides or nucleicacids commonly found associated with the polypeptide.

By the term “treat,” “treating,” or “treatment of” (or grammaticallyequivalent terms) it is meant that the severity of the subject'scondition is reduced or at least partially improved or amelioratedand/or that some alleviation, mitigation or decrease in at least oneclinical symptom is achieved and/or there is a delay in the progressionof the condition and/or prevention or delay of the onset of a disease ordisorder. The term “treat,” “treats,” “treating,” or “treatment of” andthe like also include prophylactic treatment of the subject (e.g., toprevent the onset of infection or cancer or a disorder). As used herein,the term “prevent,” “prevents,” or “prevention” (and grammaticalequivalents thereof) are not meant to imply complete abolition ofdisease and encompasses any type of prophylactic treatment that reducesthe incidence of the condition, delays the onset and/or progression ofthe condition, and/or reduces the symptoms associated with thecondition. Thus, unless the context indicates otherwise, the term“treat,” “treating,” or “treatment of” (or grammatically equivalentterms) refer to both prophylactic and therapeutic regimens.

An “effective” or “therapeutically effective” amount as used herein isan amount that is sufficient to provide some improvement or benefit tothe subject. Alternatively stated, an “effective” or “therapeuticallyeffective” amount is an amount that will provide some alleviation,mitigation, or decrease in at least one clinical symptom in the subject.Those skilled in the art will appreciate that the therapeutic effectsneed not be complete or curative, as long as some benefit is provided tothe subject.

A “heterologous nucleotide sequence” or “heterologous nucleic acid” is asequence that is not naturally occurring in the virus. Generally, theheterologous nucleic acid or nucleotide sequence comprises an openreading frame that encodes a polypeptide and/or a nontranslated RNA.

A “therapeutic polypeptide” can be a polypeptide that can alleviate orreduce symptoms that result from an absence or defect in a protein in acell or subject. In addition, a “therapeutic polypeptide” can be apolypeptide that otherwise confers a benefit to a subject, e.g.,anti-cancer effects or improvement in transplant survivability.

As used herein, the term “vector,” “virus vector,” “delivery vector”(and similar terms) generally refers to a virus particle that functionsas a nucleic acid delivery vehicle, and which comprises the viralnucleic acid (i.e., the vector genome) packaged within the virion. Virusvectors according to the present invention comprise a chimeric AAVcapsid according to the invention and can package an AAV or rAAV genomeor any other nucleic acid including viral nucleic acids. Alternatively,in some contexts, the term “vector,” “virus vector,” “delivery vector”(and similar terms) may be used to refer to the vector genome (e.g.,vDNA) in the absence of the virion and/or to a viral capsid that acts asa transporter to deliver molecules tethered to the capsid or packagedwithin the capsid.

A “recombinant AAV vector genome” or “rAAV genome” is an AAV genome(i.e., vDNA) that comprises at least one inverted terminal repeat (e.g.,one, two or three inverted terminal repeats) and one or moreheterologous nucleotide sequences. rAAV vectors generally retain the 145base terminal repeat(s) (TR(s)) in cis to generate virus; however,modified AAV TRs and non-AAV TRs including partially or completelysynthetic sequences can also serve this purpose. All other viralsequences are dispensable and may be supplied in trans (Muzyczka, (1992)Curr. Topics Microbiol. Immunol. 158:97). The rAAV vector optionallycomprises two TRs (e.g., AAV TRs), which generally will be at the 5′ and3′ ends of the heterologous nucleotide sequence(s), but need not becontiguous thereto. The TRs can be the same or different from eachother. The vector genome can also contain a single ITR at its 3′ or 5′end.

The term “terminal repeat” or “TR” includes any viral terminal repeat orsynthetic sequence that forms a hairpin structure and functions as aninverted terminal repeat (i.e., mediates the desired functions such asreplication, virus packaging, integration and/or provirus rescue, andthe like). The TR can be an AAV TR or a non-AAV TR. For example, anon-AAV TR sequence such as those of other parvoviruses (e.g., canineparvovirus (CPV), mouse parvovirus (MVM), human parvovirus B-19) or theSV40 hairpin that serves as the origin of SV40 replication can be usedas a TR, which can further be modified by truncation, substitution,deletion, insertion and/or addition. Further, the TR can be partially orcompletely synthetic, such as the “double-D sequence” as described inU.S. Pat. No. 5,478,745 to Samulski et al.

An “AAV terminal repeat” or “AAV TR” may be from any AAV, including butnot limited to serotypes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11 or anyother AAV now known or later discovered (see, e.g., Table 1). An AAVterminal repeat need not have the native terminal repeat sequence (e.g.,a native AAV TR sequence may be altered by insertion, deletion,truncation and/or missense mutations), as long as the terminal repeatmediates the desired functions, e.g., replication, virus packaging,integration, and/or provirus rescue, and the like.

The terms “rAAV particle” and “rAAV virion” are used interchangeablyhere. A “rAAV particle” or “rAAV virion” comprises a rAAV vector genomepackaged within an AAV capsid.

The AAV capsid structure is described in more detail in BERNARD N.FIELDS et al., VIROLOGY, volume 2, chapters 69 & 70 (4th ed.,Lippincott-Raven Publishers).

By “substantially retain” a property, it is meant that at least about75%, 85%, 90%, 95%, 97%, 98%, 99% or 100% of the property (e.g.,activity or other measurable characteristic) is retained.

II. Chimeric AAV Capsids Targeted to Oligodendrocytes

The inventors have identified chimeric AAV capsid structures capable ofpreferentially transducing oligodendrocytes over neurons and other cellsof the CNS. Thus, one aspect of the invention relates to a nucleic acidencoding an AAV capsid, the nucleic acid comprising, consistingessentially of, or consisting of an AAV capsid coding sequence that isat least 90% identical to: (a) the nucleotide sequence of SEQ ID NO:1;or (b) a nucleotide sequence encoding SEQ ID NO:2; and virusescomprising the chimeric AAV capsids. In some embodiments, the AAV capsidcoding sequence is at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or99% identical to the nucleotide sequence of (a) or (b). In anotherembodiment, the AAV capsid coding sequence comprises, consistessentially of, or consist of the nucleotide sequence of (a) or (b).

AAV Capsid Nucleotide Sequence of BNP61  (SEQ ID NO: 1)atggctgccg atggttatct tccagattgg ctcgaggaca ctctctctga   50aggaataaga cagtggtgga agctcaaacc tggcccacca ccaccaaagc  100ccgcagagcg gcataaggac gacagcaggg gtcttgtgct tcctgggtac  150aagtacctcg gacccttcaa cggactcgac aagggagagc cggtcaacga  200ggcagacgcc gcggccctcg agcacgacaa agcctacgac cggcagctcg  250acagcggaga caacccgtac ctcaagtaca accacgccga cgcggagttt  300caggagcgcc ttaaagaaga tacgtctttt gggggcaacc tcgggcgagc  350agtcttccag gccaaaaaga ggcttctpga acctcttggt ctggttgagg  400aagcggctaa gacggctcct ggaaagaaga ggcctgtaga gcagtctcct  450caggaaccgg actcctcctc gggcatcggc aagacaggcc agcagcccgc  500taaaaagaga ctcaatttcg gtcagactgg cgacacagag tcagtcccag  550accctcaacc aatcggagaa cctcccgcag ccccctcagg tgtgggatct  600cttacaatgg cttcaggtgg tggcgcacca gtggcagaca ataacgaagg  650tgccgatgga gtgggtagtt cctcgggaaa ttggcattgc gattcccaat  700ggctggggga cagagtcatc accaccagca cccgaacctg ggccctgccc  750acctacaaca atcacctcta caagcaaatc tccaacggga catcgggagg  800agccaccaac gacaacacct acttcggcta cagcaccccc tgggggtatt  850ttgactttaa cagattccac tgccactttt caccacgtga ctggcagcga  900ctcatcaaca acaactgggg attccggccc aagagactca gCttcaagct  950cttcaacatc caggtcaagg aggtcacgca gaatgaaggc accaagacca 1000tcgccaataa ccttaccagc acggtccagg tcttcacgga ctcggagtac 1050cagctgccgt acgttctcgg ctctgcccac cagggctgcc tgcctccgtt 1100cccggcggac gtgttcatga ttccccagta cggctaccta acactcaaca 1150acggtagtca ggccgtggga cgctcctcct tctactgcct ggaatacttt 1200ccttcgcaga tgctgagaac cggcaacaac ttccagttta cttacacctt 1250cgaggacgtg cctttccaca gcagctacgc ccacagccag agcttggacc 1300ggctgatgaa tcctctgatt gaccagtacc tgtactactt gtctcggact 1350caaacaacag gaggcacggc aaatacgcag actctgggct tcagccaagg 1400tgggcctaat acaatggcca atcaggcaaa gaactggctg ccaggaccct 1450gttaccgcca acaacgcgtc tcaacgacaa ccgggcaaaa caacaatagc 1500aactttgcct ggactgctgg gaccaaatac catctgaatg gaagaaattc 1550attggctaat cctggcatcg ctatggcaac acacaaagac gacaaggagc 1600gtttttttcc cagtaacggg atcctgattt ttggcaaaca aaatgctgcc 1650agagacaatg cggattacag cgatgtcatg ctcaccagcg aggaagaaat 1700caaaaccact aaccctgtgg ctacagagga atacggtatc gtggcagata 1750acttgcagca gcaaaacacg gctcctcaaa ttggaactgt caacagccag 1800ggggccttac ccggtatggt ttggcagaac cgggacgtgt acctgcaggg 1850tcccatgtgg gccaagattc ctcacacgga cggcaacttc cacccgtctc 1900cgctgatggg cggctttggc ctgaaacatc ctccgcctca gatcctgatc 1950aagaacacgc ctgtacctgc ggatcctccg accaccttca accagtcaaa 20C0gctgaactct ttcatcacgc aatacagcac cggacaggtc agcgtggaaa 2050ctgaatggga gctgcagaag gaaaacagca agcgctggaa ccccgagatc 2100cagtacacct ccaactacta caaatctaca agtgtggact ttgctgttaa 2150tacagaaggc gtgtactctg aaccccaccc cattggcacc cgttacctca 2200cccgtcccct gtaa AAV Capsid Amino Acid Sequence of BNP61 (SEQ ID NO: 2)MAADGYLPDW LEDTLSEGIR QWWKLKPGPP PPKPAERHKD DSRGLVLPGY  50KYLGPFNGLD KGEPVNEADA AALEHDKAYD RQLDSGDNPY LKYNHADAEF 100QERLQGDTSF GGNLGRAVFQ AKKRVLEPLG LVEEAAKTAP GKKRPVEQSP 150QEPDSSSGIG ETGQQPAKKR LNFGQTGDSE SVPDPQPLGE PPATPAAVGP 200TTMASGGGAP MADNNEGADG VGSSSGNWHC DSQWLGDRVI TTSTRTWALP 250TYNNHLYKQI SSASTGASND NHYFGYSTPW GYFDFNRFHC HFSPRDWQRL 300INNNWGFRPK RLSFKLFNIQ VKEVTDNNGV KTIANNLTST VQVFTDSEYQ 350LPYVLGSAHQ GCLPPFPADV FMIPQYGYLT LNNGSQAVGR SSFYCLEYFP 400SQMLRTGNNF TFSYTFEDVP FHSSYAHSQS LDRLMNPLID QYLYYLSRTQ 450TTGGTANTQT LGFSQGGPNT MANQAKNWLP GPCYRQQRVS TTTGQNNNSN 500FAWTAGTKYH LNGRNSLANP GIAMATHKDD KERFFPSNGI LIFGKQNAAR 550DNADYSDVML TSEEEIKTTN PVATEEYGIV ADNLQQQNTA PQIGTVNSQG 600ALPGMVWQNR DVYLQGPIWA KIPHTDGNFH PSPLMGGFGL KHPPPQILIK 650NTPVPADPPT TFNQSKLNSF ITQYSTGQVS VEIEWELQKE NSKRWNPEIQ 700YTSNYYKSTS VDFAVNTEGV YSEPHPIGTR YLTRPL

In some embodiments, the nucleic acid encoding an AAV capsid comprises,consists essentially of, or consists of an AAV capsid coding sequencethat is at least 90% identical to a nucleotide sequence encoding SEQ IDNOS:3 or 4; and viruses comprising the chimeric AAV capsids. In someembodiments, the AAV capsid coding sequence is at least 91%, 92%, 93%,94%, 95%, 96%, 97%, 98% or 99% identical to the nucleotide sequenceencoding SEQ ID NOS:3 or 4. In another embodiment, the AAV capsid codingsequence comprises, consists essentially of, or consists of thenucleotide sequence encoding SEQ ID NOS:3 or 4.

AAV Capsid Amino Acid Sequence of BNP62 (SEQ ID NO: 3)MAADGYLPDW LEDTLSEGIR QWWKLKPGPP PPKPAERHKD DSRGLVLPGY  50KYLGPFNGLD KGEPVNEADA AALEHDKAYD RQLDSGDNPY LKYNHADAEF 100QERLQGDTSF GGNLGRAVFQ AKKRVLEPLG LVEEAAKTAP GKKRPVEQSP 150QEPDSSSGIG ETGQQPAKKR LNFGQTGDSE SVPDPQPLGE PPATPAAVGP 200TTMASGGGAP MADNNEGADG VGSSSGNWHC DSQWLGDRVI TTSTRTWALP 250TYNNHLYKQI SSASTGASND NHYFGYSTPW GYFDFNRFHC HFSPRDWQRL 300INNNWGFRPK RLSFKLFNIQ VKEVTDNNGV KTIANNLTST VQVFTDSEYQ 350LPYVLGSAHQ GCLPPFPADV FMIPQYGYLT LNNGSQAVGR SSFYCLEYFP 400SQMLRTGNNF TFSYTFEDVP FHSSYAHSQS LDRLMNPLID QYLYYLSRTQ 450TTGGTANTQT LGFSQGGPNT MANQAKNWLP GPCYRQQRVS TTTGQNNNSN 500FAWTAGTKYH LNGRNSLANP GIAMATHKDD KERFFPSNGI LIFGKQNAAR 550DNADYSDVML TSEEEIKTTN PVATEEYGIV ADNLQQQNTA PQIGTVNSQG 600ALPGMVWQNR DVYLQGPIWA KIPHTDGNFH PSPLMGGFGL KHPPPQILIK 650NTPVPADPPT TFNQSKLNSF ITQYSTGQVS VEIEWELQKE NSKRWNPEIQ 700YTSNYYKSTS VDFAVNTEGV YSEPHPIGTR YLTRPLAAV Capsid Amino Acid Sequence of BNP63 (SEQ ID NO: 4)MAADGYLPDW LEDTLSEGIR QWWKLKPGPP PPKPAERHKD DSRGLVLPGY  50KYLGPFNGLD KGEPVNEADA AALEHDKAYD RQLDSGDNPY LKYNHADAEF 100QERLQGDTSF GGNLGRAVFQ AKKRVLEPLG LVEEAAKTAP GKKRPVEQSP 150QEPDSSSGIG ETGQQPAKKR LNFGQTGDSE SVPDPQPLGE PPATPAAVGP 200TTMASGGGAP MADNNEGADG VGSSSGNWHC DSQWLGDRVI TTSTRTWALP 250TYNNHLYKQI SSASTGASND NHYFGYSTPW GYFDFNRFHC HFSPRDWQRL 300INNNWGFRPK RLSFKLFNIQ VKEVTDNNGV KTIANNLTST VQVFTDSEYQ 350LPYVLGSAHQ GCLPPFPADV FMIPQYGYLT LNNGSQAVGR SSFYCLEYFP 400SQMLRTGNNF TFSYTFEDVP FHSSYAHSQS LDRLMNPLID QYLYYLSRTQ 450TTGGTANTQT LGFSQGGPNT MANQAKNWLP GPCYRQQRVS TTTGQNNNSN 500FAWTAGTKYH LNGRNSLANP GIAMATHKDD KERFFPSNGI LIFGKQNAAR 550DNADYSDVML TSEEEIKTTN PVATEEYGIV ADNLQQQNTA PQIGTVNSQG 600ALPGMVWQNR DVYLQGPIWA KIPHTDGNFH PSPLMGGFGL KHPPPQILIK 650NTPVPADPPT TFNQSKLNSF ITQYSTGQVS VEIEWELQKE NSKRWNPEIQ 700YTSNYYKSTS VDFAVNTEGV YSEPHPIGTR YLTRPL

SEQ ID NOS:2-4 show examples of the VP1 capsid protein sequences of theinvention. The designation of all amino acid positions in thedescription of the invention and the appended claims is with respect toVP1 numbering. Those skilled in the art will understand that the AAVcapsid generally contains the smaller VP2 and VP3 capsid proteins aswell. Due to the overlap of the coding sequences for the AAV capsidproteins, the nucleic acid coding sequences and amino acid sequences ofthe VP2 and VP3 capsid proteins will be apparent from the VP1 sequencesshown in SEQ ID NOS:1-4. In particular, VP2 starts at nucleotide 412(acg) of SEQ ID NO:1 and threonine 148 of SEQ ID NO:2. VP3 starts atnucleotide 607 (atg) of SEQ ID NO:1 and methionine 203 of SEQ ID NO:2.In certain embodiments, isolated VP2 and VP3 capsid proteins comprisingthe sequence from SEQ ID NOS:2-4 and isolated nucleic acids encoding theVP2 or VP3 proteins, or both, are contemplated.

The invention also provides chimeric AAV capsid proteins and chimericcapsids, wherein the capsid protein comprises, consists essentially of,or consists of an amino acid sequence as shown in one of SEQ ID NOS:2-4,wherein 1, 2 or fewer, 3 or fewer, 4 or fewer, 5 or fewer, 6 or fewer, 7or fewer, 8 or fewer, 9 or fewer, 10 or fewer, 12 or fewer, 15 or fewer,20 or fewer, 25 or fewer, 30 or fewer, 40 or fewer, or 50 or fewer ofthe amino acids within the capsid protein coding sequence of one of SEQID NOS:2-4 is substituted by another amino acid (naturally occurring,modified and/or synthetic), optionally a conservative amino acidsubstitution, and/or are deleted and/or there are insertions (includingN-terminal and C-terminal extensions) of 1, 2 or fewer, 3 or fewer, 4 orfewer, 5 or fewer, 6 or fewer, 7 or fewer, 8 or fewer, 9 or fewer, 10 orfewer, 12 or fewer, 15 or fewer, 20 or fewer, 25 or fewer, 30 or fewer,40 or fewer, or 50 or fewer amino acids or any combination ofsubstitutions, deletions and/or insertions, wherein the substitutions,deletions and/or insertions do not unduly impair the structure and/orfunction of a virion (e.g., an AAV virion) comprising the variant capsidprotein or capsid. For example, in representative embodiments of theinvention, an AAV virion comprising the chimeric capsid proteinsubstantially retains at least one property of a chimeric virioncomprising a chimeric capsid protein as shown in one of SEQ ID NOS:2-4.For example, the virion comprising the chimeric capsid protein cansubstantially retain the oligodendrocyte tropism profile of a virioncomprising the chimeric AAV capsid protein as shown in one of SEQ IDNOS:2-4. Methods of evaluating biological properties such as virustransduction are well-known in the art (see, e.g., the Examples).

Conservative amino acid substitutions are known in the art. Inparticular embodiments, a conservative amino acid substitution includessubstitutions within one or more of the following groups: glycine,alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid;asparagine, glutamine; serine, threonine; lysine, arginine; and/orphenylalanine, tyrosine.

It will be apparent to those skilled in the art that the amino acidsequences of the chimeric AAV capsid protein of SEQ ID NOS:2-4 canfurther be modified to incorporate other modifications as known in theart to impart desired properties. As nonlimiting possibilities, thecapsid protein can be modified to incorporate targeting sequences (e.g.,ROD) or sequences that facilitate purification and/or detection. Forexample, the capsid protein can be fused to all or a portion ofglutathione-S-transferase, maltose-binding protein, a heparin/heparansulfate binding domain, poly-His, a ligand, and/or a reporter protein(e.g., Green Fluorescent Protein, β-glucuronidase, β-galactosidase,luciferase, etc), an immunoglobulin Fc fragment, a single-chainantibody, hemagglutinin, c-myc, FLAG epitope, and the like to form afusion protein. Methods of inserting targeting peptides into the AAVcapsid are known in the art (see, e.g., international patent publicationWO 00/28004; Nicklin et at, (2001) Mol. Ther. 474-181; White et al.,(2004) Circulation 109:513-319; Muller et al., (2003) Nature Biotech.21:1040-1046.

The viruses of the invention can further comprise a duplexed viralgenome as described in international patent publication WO 01/92551 andU.S. Pat. No. 7,465,583.

The invention also provides AAV capsids comprising the chimeric AAVcapsid proteins of the invention and virus particles (i.e., virions)comprising the same, wherein the virus particle packages (i.e.,encapsidates) a vector genome, optionally an AAV vector genome. Inparticular embodiments, the invention provides an AAV particlecomprising an AAV capsid comprising an AAV capsid protein of theinvention, wherein the AAV capsid packages an AAV vector genome. Theinvention also provides an AAV particle comprising an AAV capsid or AAVcapsid protein encoded by the chimeric nucleic acid capsid codingsequences of the invention.

In particular embodiments, the virion is a recombinant vector comprisinga heterologous nucleic acid of interest, e.g., for delivery to a cell.Thus, the present invention is useful for the delivery of nucleic acidsto cells in vitro, ex vivo, and in vivo. In representative embodiments,the recombinant vector of the invention can be advantageously employedto deliver or transfer nucleic acids to animal (e.g., mammalian) cells.

Any heterologous nucleotide sequence(s) may be delivered by a virusvector of the present invention. Nucleic acids of interest includenucleic acids encoding polypeptides, optionally therapeutic (e.g., formedical or veterinary uses) and/or immunogenic (e.g., for vaccines)polypeptides.

In some embodiments, the polypeptide is one that stimulates growthand/or differentiation of oligodendrocytes. Examples include, withoutlimitation, insulin-like growth factor-1, glial-derived neurotrophicfactor, neurotrophin-3, artemin, transforming growth factor alpha,platelet-derived growth factor, leukemia inhibitory factor, prolactin,monocarboxylate transporter 1, or nuclear factor 1A.

Therapeutic polypeptides include, but are not limited to, cysticfibrosis transmembrane regulator protein (CFTR), dystrophin (includingthe protein product of dystrophin mini-genes or micro-genes, see, e.g.,Vincent et al., (1993) Nature Genetics 5:130; U.S. Patent PublicationNo. 2003017131; Wang et al., (2000) Proc. Natl. Acad. Sci. USA97:13714-9 [mini-dystrophin]; Harper et al., (2002) Nature Med. 8:253-61[micro-dystrophin]); mini-agrin, a laminin-α2, a sarcoglycan (α, β, γ orδ), Fukutin-related protein, myostatin pro-peptide, follistatin,dominant negative myostatin, an angiogenic factor (e.g., VEGF,angiopoietin-1 or 2), an anti-apoptotic factor (e.g., heme-oxygenase-1,TGF-β, inhibitors of pro-apoptotic signals such as caspases, proteases,kinases, death receptors [e.g., CD-095], modulators of cytochrome Crelease, inhibitors of mitochondrial pore opening and swelling); activintype II soluble receptor, anti-inflammatory polypeptides such as theIkappa B dominant mutant, sarcospan, utrophin, mini-utrophin, antibodiesor antibody fragments against myostatin or myostatin propeptide, cellcycle modulators, Rho kinase modulators such as Cethrin, which is amodified bacterial C3 exoenzyme [available from BioAxone Therapeutics,Inc., Saint-Lauren, Quebec, Canada], BCL-xL, BCL2, XIAP, FLICEc-s,dominant-negative caspase-8, dominant negative caspase-9, SPI-6 (see,e.g., U.S. Patent Application No. 20070026076), transcriptional factorPGC-α1, Pinch gene, ILK gene and thymosin 34 gene), clotting factors(e.g., Factor VIII, Factor IX, Factor X, etc.), erythropoietin,angiostatin, endostatin, catalase, tyrosine hydroxylase, anintracellular and/or extracellular superoxide dismutase, leptin, the LDLreceptor, neprilysin, lipoprotein lipase, ornithine transcarbamylase,β-globin, α-globin, spectrin, α₁-antitrypsin, adenosine deaminase,hypoxanthine guanine phosphoribosyl transferase, β-glucocerebrosidase,sphingomyelinase, lysosomal hexosaminidase A, branched-chain keto aciddehydrogenase, RP65 protein, a cytokine (e.g., α-interferon,β-interferon, interferon-γ, interleukins-1 through-14,granulocyte-macrophage colony stimulating factor, lymphotoxin, and thelike), peptide growth factors, neurotrophic factors and hormones (e.g.,somatotropin, insulin, insulin-like growth factors including IGF-1 andIGF-2, GLP-1, platelet derived growth factor, epidermal growth factor,fibroblast growth factor, nerve growth factor, neurotrophic factor-3 and-4, brain-derived neurotrophic factor, glial derived growth factor,transforming growth factor-α and -β, and the like), bone morphogenicproteins (including RANKL and VEGF), a lysosomal protein, a glutamatereceptor, a lymphokine, soluble CD4, an Fe receptor, a T cell receptor,ApoE, ApoC, inhibitor 1 of protein phosphatase inhibitor 1 (I-1),phospholamban, serca2a, lysosomal acid α-glucosidase, α-galactosidase A,Barket, β2-adrenergic receptor, β2-adrenergic receptor kinase (BARK),phosphoinositide-3 kinase (PI3 kinase), calsarcin, a receptor (e.g., thetumor necrosis growth factor-α soluble receptor), an anti-inflammatoryfactor such as IRAP, Pim-1, PGC-1α, SOD-1, SOD-2, ECF-SOD, kallikrein,thymosin-β4, hypoxia-inducible transcription factor [HIF], an angiogenicfactor, S100A1, parvalbumin, adenylyl cyclase type 6, a molecule thateffects G-protein coupled receptor kinase type 2 knockdown such as atruncated constitutively active bARKct; phospholamban inhibitory ordominant-negative molecules such as phospholamban S16E, a monoclonalantibody (including single chain monoclonal antibodies) or a suicidegene product (e.g., thymidine kinase, cytosine deaminase, diphtheriatoxin, and tumor necrosis factors such as TNF-α), and any otherpolypeptide that has a therapeutic effect in a subject in need thereof.

Heterologous nucleotide sequences encoding polypeptides include thoseencoding reporter polypeptides (e.g., an enzyme). Reporter polypeptidesare known in the art and include, but are not limited to, GreenFluorescent Protein, β-galactosidase, alkaline phosphatase, luciferase,and chloramphenicol acetyltransferase.

Alternatively, the heterologous nucleic acid may encode an antisenseoligonucleotide, a ribozyme (e.g., as described in U.S. Pat. No.5,877,022), RNAs that effect spliceosome-mediated trans-splicing (see,Puttaerju et al., (1999) Nature Biotech. 17:246; U.S. Pat. No.6,013,487; U.S. Pat. No. 6,083,702), interfering RNAs (RNAi) includingsmall interfering RNAs (siRNA) that mediate gene silencing (see, Sharpet al., (2000) Science 287:2431), microRNA, or other non-translated“functional” RNAs, such as “guide” RNAs (Gorman et al., (1998) Proc.Nat. Acad. Sci. USA 95:4929; U.S. Pat. No. 5,869,248 to Yuan et al.),and the like. Exemplary untranslated RNAs include RNAi or antisense RNAagainst the multiple drug resistance (MDR) gene product (e.g., to treattumors and/or for administration to the heart to prevent damage bychemotherapy), RNAi or antisense RNA against myostatin (Duchenne orBecker muscular dystrophy), RNAi or antisense RNA against VEGF or atumor immunogen including but not limited to those tumor immunogensspecifically described herein (to treat tumors), RNAi or antisenseoligonucleotides targeting mutated dystrophins (Duchenne or Beckermuscular dystrophy), RNAi or antisense RNA against the hepatitis Bsurface antigen gene (to prevent and/or treat hepatitis B infection),RNAi or antisense RNA against the HIV tat and/or rev genes (to preventand/or treat HIV) and/or RNAi or antisense RNA against any otherimmunogen from a pathogen (to protect a subject from the pathogen) or adefective gene product (to prevent or treat disease). RNAi or antisenseRNA against the targets described above or any other target can also beemployed as a research reagent.

As is known in the art, anti-sense nucleic acids (e.g., DNA or RNA) andinhibitory RNA (e.g., microRNA and RNAi such as siRNA or shRNA)sequences can be used to induce “exon skipping” in patients withmuscular dystrophy arising from defects in the dystrophin gene. Thus,the heterologous nucleic acid can encode an antisense nucleic acid orinhibitory RNA that induces appropriate exon skipping. Those skilled inthe art will appreciate that the particular approach to exon skippingdepends upon the nature of the underlying defect in the dystrophin gene,and numerous such strategies are known in the art. Exemplary antisensenucleic acids and inhibitory RNA sequences target the upstream branchpoint and/or downstream donor splice site and/or internal splicingenhancer sequence of one or more of the dystrophin exons (e.g., exons 19or 23). For example, in particular embodiments, the heterologous nucleicacid encodes an antisense nucleic acid or inhibitory RNA directedagainst the upstream branch point and downstream splice donor site ofexon 19 or 23 of the dystrophin gene. Such sequences can be incorporatedinto an AAV vector delivering a modified U7 snRNA and the antisensenucleic acid or inhibitory RNA (see, e.g., Goyenvalle et al., (2004)Science 306:1796-1799). As another strategy, a modified U1 snRNA can beincorporated into an AAV vector along with siRNA, microRNA or antisenseRNA complementary to the upstream and downstream splice sites of adystrophin exon (e.g., exon 19 or 23) (see, e.g., Denti et al., (2006)Proc. Nat. Acad. Sci. USA 103:3758-3763). Further, antisense nucleicacids and inhibitory RNA can target the splicing enhancer sequenceswithin exons 19, 43, 45 or 53 (see, e.g., U.S. Pat. No. 6,653,467; U.S.Pat. No. 6,727,355; and U.S. Pat. No. 6,653,466).

Ribozymes are RNA-protein complexes that cleave nucleic acids in asite-specific fashion. Ribozymes have specific catalytic domains thatpossess endonuclease activity (Kim et al., (1987) Proc. Nat. Acad. Sci.USA 84; 8788; Gerlach et al., (1987) Nature 328:802; Forster and Symons,(1987) Cell 49:211). For example, a large number of ribozymes acceleratephosphoester transfer reactions with a high degree of specificity, oftencleaving only one of several phosphoesters in an oligonucleotidesubstrate (Michel and Westhof, (1990), J. Mol. Biol. 216:585;Reinhold-Hurek and Shub, (1992) Nature 357:173). This specificity hasbeen attributed to the requirement that the substrate bind via specificbase-pairing interactions to the internal guide sequence (“IGS”) of theribozyme prior to chemical reaction.

Ribozyme catalysis has primarily been observed as part ofsequence-specific cleavage/ligation reactions involving nucleic acids(Joyce, (1989) Nature 338:217). For example, U.S. Pat. No. 5,354,855reports that certain ribozymes can act as endonucleases with a sequencespecificity greater than that of known ribonucleases and approachingthat of the DNA restriction enzymes. Thus, sequence-specificribozyme-mediated inhibition of nucleic acid expression may beparticularly suited to therapeutic applications (Scanlon et al., (1991)Proc. Natl. Acad. Sci. USA 88:10591; Sarver et al., (1990) Science247:1222; Sioud et al., (1992) J. Mol. Biol. 223:831).

MicroRNAs (mir) are natural cellular RNA molecules that can regulate theexpression of multiple genes by controlling the stability of the mRNA.Over-expression or diminution of a particular microRNA can be used totreat a dysfunction and has been shown to be effective in a number ofdisease states and animal models of disease (see, e.g., Couzin, (2008)Science 319:1782-4). The chimeric AAV can be used to deliver microRNAinto cells, tissues and subjects for the treatment of genetic andacquired diseases, or to enhance functionality and promote growth ofcertain tissues. For example, mir-1, mir-133, mir-206 and/or mir-208 canbe used to treat cardiac and skeletal muscle disease (see, e.g., Chen etal., (2006) Genet. 38:228-33; van Rooij et al., (2008) Trends Genet. 24;159-66). MicroRNA can also be used to modulate the immune system aftergene delivery (Brown et at, (2007) Blood 110:4144-52).

The term “antisense oligonucleotide” (including “antisense RNA”) as usedherein, refers to a nucleic acid that is complementary to andspecifically hybridizes to a specified DNA or RNA sequence. Antisenseoligonucleotides and nucleic acids that encode the same can be made inaccordance with conventional techniques. See, e.g., U.S. Pat. No.5,023,243 to Tullis; U.S. Pat. No. 5,149,797 to Pederson et a.

Those skilled in the art will appreciate that it is not necessary thatthe antisense oligonucleotide be fully complementary to the targetsequence as long as the degree of sequence similarity is sufficient forthe antisense nucleotide sequence to specifically hybridize to itstarget (as defined above) and reduce production of the protein product(e.g., by at least about 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% ormore).

To determine the specificity of hybridization, hybridization of sucholigonucleotides to target sequences can be carried out under conditionsof reduced stringency, medium stringency or even stringent conditions.Suitable conditions for achieving reduced, medium and stringenthybridization conditions are as described herein.

Alternatively stated, in particular embodiments, antisenseoligonucleotides of the invention have at least about 60%, 70%, 80%,90%, 95%, 97%, 98% or higher sequence identity with the complement ofthe target sequence and reduce production of the protein product (asdefined above). In some embodiments, the antisense sequence contains 1,2, 3, 4, 5, 6, 7, 8, 9 or 10 mismatches as compared with the targetsequence.

Methods of determining percent identity of nucleic acid sequences aredescribed in more detail elsewhere herein.

The length of the antisense oligonucleotide is not critical as long asit specifically hybridizes to the intended target and reduces productionof the protein product (as defined above) and can be determined inaccordance with routine procedures. In general, the antisenseoligonucleotide is at least about eight, ten or twelve or fifteennucleotides in length and/or less than about 20, 30, 40, 50, 60, 70, 80,100 or 150 nucleotides in length.

RNA interference (RNAi) is another useful approach for reducingproduction of a protein product (e.g., shRNA or siRNA). RNAi is amechanism of post-transcriptional gene silencing in whichdouble-stranded RNA (dsRNA) corresponding to a target sequence ofinterest is introduced into a cell or an organism, resulting indegradation of the corresponding mRNA. The mechanism by which RNAiachieves gene silencing has been reviewed in Sharp et al., (2001) GenesDev 15: 485-490; and Hammond et al., (2001) Nature Rev. Gen. 2:110-119).The RNAi effect persists for multiple cell divisions before geneexpression is regained. RNAi is therefore a powerful method for makingtargeted knockouts or “knockdowns” at the RNA level. RNAi has provensuccessful in human cells, including human embryonic kidney and HeLacells (see, e.g., Elbashir et al., Nature (2001) 411:494-8).

Initial attempts to use RNAi in mammalian cells resulted in antiviraldefense mechanisms involving PKR in response to the dsRNA molecules(see, e.g., Gil et al., (2000) Apoptosis 5:107). It has since beendemonstrated that short synthetic dsRNA of about 21 nucleotides, knownas “short interfering RNAs” (siRNA) can mediate silencing in mammaliancells without triggering the antiviral response (see, e.g., Elbashir etal., Nature (2001) 411:494-8; Caplen et al., (2001) Proc. Nat. Acad.Sci. USA 98; 9742).

The RNAi molecule (including an siRNA molecule) can be a short hairpinRNA (shRNA; see Paddison et al., (2002), Proc. Nat. Acad. Sci. USA99:1443-1448), which is believed to be processed in the cell by theaction of the RNase III like enzyme Dicer into 20-25mer siRNA molecules.The shRNAs generally have a stem-loop structure in which two invertedrepeat sequences are separated by a short spacer sequence that loopsout. There have been reports of shRNAs with loops ranging from 3 to 23nucleotides in length. The loop sequence is generally not critical.Exemplary loop sequences include the following motifs: AUG, CCC, UUCG,CCACC, CTCGAG, AAGCUU, CCACACC and UUCAAGAGA.

The RNAi can further comprise a circular molecule comprising sense andantisense regions with two loop regions on either side to form a“dumbbell” shaped structure upon dsRNA formation between the sense andantisense regions. This molecule can be processed in vitro or in vivo torelease the dsRNA portion, e.g., a siRNA.

International patent publication WO 01/77350 describes a vector forbi-directional transcription to generate both sense and antisensetranscripts of a heterologous sequence in a eukaryotic cell. Thistechnique can be employed to produce RNAi for use according to theinvention.

Shinagawa et al., (2003) Genes Dev. 17:1340 reported a method ofexpressing long dsRNAs from a CMV promoter (a pol II promoter), whichmethod is also applicable to tissue specific pol II promoters. Likewise,the approach of Xia at al., (2002) Nature Biotech. 20:1006, avoidspoly(A) tailing and can be used in connection with tissue-specificpromoters.

Methods of generating RNAi include chemical synthesis, in vitrotranscription, digestion of long dsRNA by Dicer (in vitro or in vivo),expression in vivo from a delivery vector, and expression in vivo from aPCR-derived RNAi expression cassette (see, e.g., TechNotes 10(3) “FiveWays to Produce siRNAs,” from Ambion. Inc., Austin Tex.; available atwww.ambion.com).

Guidelines for designing siRNA molecules are available (see e.g.,literature from Ambion, Inc., Austin Tex.; available at www.ambion.com).In particular embodiments, the siRNA sequence has about 30-50% 0/Ccontent. Further, long stretches of greater than four T or A residuesare generally avoided if RNA polymerase III is used to transcribe theRNA. Online siRNA target finders are available, e.g., from Ambion, Inc.(www.ambion.com), through the Whitehead Institute of Biomedical Research(www.jura.wi.mit.edu) or from Dharmacon Research, Inc.(www.dharmacon.com).

The antisense region of the RNAi molecule can be completelycomplementary to the target sequence, but need not be as long as itspecifically hybridizes to the target sequence (as defined above) andreduces production of the protein product (e.g., by at least about 30%,40%, 50%, 60%, 70%, 80%, 90%, 95% or more). In some embodiments,hybridization of such oligonucleotides to target sequences can becarried out under conditions of reduced stringency, medium stringency oreven stringent conditions, as defined above.

In other embodiments, the antisense region of the RNAi has at leastabout 60%, 70%, 80%, 90%, 95%, 97%, 98% or higher sequence identity withthe complement of the target sequence and reduces production of theprotein product (e.g., by at least about 30%, 40%, 50%, 60%, 70%, 80%,90%, 95% or more). In some embodiments, the antisense region contains 1,2, 3, 4, 5, 6, 7, 8, 9 or 10 mismatches as compared with the targetsequence. Mismatches are generally tolerated better at the ends of thedsRNA than in the center portion.

In particular embodiments, the RNAi is formed by intermolecularcomplexing between two separate sense and antisense molecules. The RNAicomprises a double stranded region formed by the intermolecularbasepairing between the two separate strands. In other embodiments, theRNAi comprises a ds region formed by intramolecular basepairing within asingle nucleic acid molecule comprising both sense and antisenseregions, typically as an inverted repeat (e.g., a shRNA or other stemloop structure, or a circular RNAi molecule). The RNAi can furthercomprise a spacer region between the sense and antisense regions.

Generally, RNAi molecules are highly selective. If desired, thoseskilled in the art can readily eliminate candidate RNAi that are likelyto interfere with expression of nucleic acids other than the target bysearching relevant databases to identify RNAi sequences that do not havesubstantial sequence homology with other known sequences, for example,using BLAST (available at www.ncbi.nlm.nih.gov/BLAST).

Kits for the production of RNAi are commercially available, e.g., fromNew England Biolabs, Inc. and Ambion, Inc.

The recombinant virus vector may also comprise a heterologous nucleotidesequence that shares homology with and recombines with a locus on thehost chromosome. This approach may be utilized to correct a geneticdefect in the host cell.

The present invention also provides recombinant virus vectors thatexpress an immunogenic polypeptide, e.g., for vaccination. Theheterologous nucleic acid may encode any immunogen of interest known inthe art including, but are not limited to, immunogens from humanimmunodeficiency virus, influenza virus, gag proteins, tumor antigens,cancer antigens, bacterial antigens, viral antigens, and the like.Alternatively, the immunogen can be presented in the virus capsid (e.g.,incorporated therein) or tethered to the virus capsid (e.g., by covalentmodification).

The use of parvoviruses as vaccines is known in the art (see, e.g.,Miyamura et at, (1994) Proc. Nat. Acad. Sci. USA 91:8507; U.S. Pat. No.5,916,563 to Young er al., U.S. Pat. No. 5,905,040 to Mazzara et al.,U.S. Pat. No. 5,882,652, U.S. Pat. No. 5,863,541 to Samulski et al; thedisclosures of which are incorporated herein in their entireties byreference). The antigen may be presented in the virus capsid.Alternatively, the antigen may be expressed from a heterologous nucleicacid introduced into a recombinant vector genome.

An immunogenic polypeptide, or immunogen, may be any polypeptidesuitable for protecting the subject against a disease, including but notlimited to microbial, bacterial, protozoal, parasitic, fungal and viraldiseases. For example, the immunogen may be an orthomyxovirus immunogen(e.g., an influenza virus immunogen, such as the influenza virushemagglutinin (HA) surface protein or the influenza virus nucleoproteingene, or an equine influenza virus immunogen), or a lentivirus immunogen(e.g., an equine infectious anemia virus immunogen, a SimianImmunodeficiency Virus (SIV) immunogen, or a Human ImmunodeficiencyVirus (HIV) immunogen, such as the HIV or SIV envelope GP160 protein,the HIV or SIV matrix/capsid proteins, and the HIV or SIV gag, pol andenv genes products). The immunogen may also be an arenavirus immunogen(e.g., Lassa fever virus immunogen, such as the Lassa fever virusnucleocapsid protein gene and the Lassa fever envelope glycoproteingene), a poxvirus immunogen (e.g., vaccinia, such as the vaccinia L1 orL8 genes), a flavivirus immunogen (e.g., a yellow fever virus immunogenor a Japanese encephalitis virus immunogen), a filovirus immunogen(e.g., an Ebola virus immunogen, or a Marburg virus immunogen, such asNP and GP genes), a bunyavirus immunogen (e.g., RVFV, CCHF, and SFSviruses), or a coronavirus immunogen (e.g., an infectious humancoronavirus immunogen, such as the human coronavirus envelopeglycoprotein gene, or a porcine transmissible gastroenteritis virusimmunogen, or an avian infectious bronchitis virus immunogen, or asevere acute respiratory syndrome (SARS) immunogen such as a S [S1 orS2], M, E, or N protein or an immunogenic fragment thereof). Theimmunogen may further be a polio immunogen, herpes immunogen (e.g., CMV,EBV, HSV immunogens) mumps immunogen, measles immunogen, rubellaimmunogen, diphtheria toxin or other diphtheria immunogen, pertussisantigen, hepatitis (e.g., hepatitis A, hepatitis B or hepatitis C)immunogen, or any other vaccine immunogen known in the art.

Alternatively, the immunogen may be any tumor or cancer cell antigen.Optionally, the tumor or cancer antigen is expressed on the surface ofthe cancer cell. Exemplary cancer and tumor cell antigens are describedin S. A. Rosenberg, (1999) Immunity 10:281). Illustrative cancer andtumor antigens include, but are not limited to: BRCA1 gene product,BRCA2 gene product, gp100, tyrosinase, GAGE-1/2, BAGE, RAGE, NY-ESO-1,CDK-4, β-catenin, MUM-1, Caspase-8, KIAA0205, HPVE, SART-1, PRAME, p15,melanoma tumor antigens (Kawakami et al., (1994) Proc. Natl. Acad. Sci.USA 91:3515; Kawakami et al., (1994) J. Exp. Med., 180:347; Kawakami etal., (1994) Cancer Res. 54:3124) including MART-1 (Coulie et al., (1991)J. Exp. Med. 180:35), gp100 (Wick et al., (1988) J. Cutan. Pathol.4:201) and MAGE antigen (MAGE-1, MAGE-2 and MAGE-3) (Van der Bruggen etal., (1991) Science, 254:1643), CEA, TRP-1; TRP-2; P-15 and tyrosinase(Brichard et al., (1993) J. Exp. Med. 178:489); HER-2/neu gene product(U.S. Pat. No. 4,968,603); CA 125; HE4; LK26; FB5 (endosialin); TAG 72;AFP; CA19-9; NSE; DU-PAN-2; CA50; Span-1; CA72-4; HCG; STN (sialyl Tnantigen); c-erbB-2 proteins; PSA; L-CanAg; estrogen receptor; milk fatglobulin; p53 tumor suppressor protein (Levine, (1993) Ann. Rev.Biochem. 62:623); mucin antigens (international patent publication WO90/05142); telomerases; nuclear matrix proteins; prostatic acidphosphatase; papilloma virus antigens; and antigens associated with thefollowing cancers: melanomas, adenocarcinoma, thymoma, sarcoma, lungcancer, liver cancer, colorectal cancer, non-Hodgkin's lymphoma,Hodgkin's lymphoma, leukemias, uterine cancer, breast cancer, prostatecancer, ovarian cancer, cervical cancer, bladder cancer, kidney cancer,pancreatic cancer, brain cancer, kidney cancer, stomach cancer,esophageal cancer, head and neck cancer and others (see, e.g.,Rosenberg, (1996) Annu. Rev. Med. 47:481-91).

Alternatively, the heterologous nucleotide sequence may encode anypolypeptide that is desirably produced in a cell in vitro, ex vivo, orin vivo. For example, the virus vectors may be introduced into culturedcells and the expressed protein product isolated therefrom.

It will be understood by those skilled in the art that the heterologousnucleic acid(s) of interest may be operably associated with appropriatecontrol sequences. For example, the heterologous nucleic acid may beoperably associated with expression control elements, such astranscription/translation control signals, origins of replication,polyadenylation signals, internal ribosome entry sites (IRES),promoters, enhancers, and the like.

Those skilled in the art will further appreciate that a variety ofpromoter/enhancer elements may be used depending on the level andtissue-specific expression desired. The promoter/enhancer may beconstitutive or inducible, depending on the pattern of expressiondesired. The promoter/enhancer may be native or foreign and can be anatural or a synthetic sequence. By foreign, it is intended that thetranscriptional initiation region is not found in the wild-type hostinto which the transcriptional initiation region is introduced.

Promoter/enhancer elements can be native to the target cell or subjectto be treated and/or native to the heterologous nucleic acid sequence.The promoter/enhancer element is generally chosen so that it willfunction in the target cell(s) of interest. In representativeembodiments, the promoter/enhancer element is a mammalianpromoter/enhancer element. The promoter/enhance element may beconstitutive or inducible.

Inducible expression control elements are generally used in thoseapplications in which it is desirable to provide regulation overexpression of the heterologous nucleic acid sequence(s). Induciblepromoters/enhancer elements for gene delivery can be tissue-specific ortissue-preferred promoter/enhancer elements, and include muscle specificor preferred (including cardiac, skeletal and/or smooth muscle), neuraltissue specific or preferred (including brain-specific), eye (includingretina-specific and cornea-specific), liver specific or preferred, bonemarrow specific or preferred, pancreatic specific or preferred, spleenspecific or preferred, and lung specific or preferred promoter/enhancerelements. In one embodiment, an oligodendrocyte-specified oroligodendrocyte-preferred promoter is used. Examples include, withoutlimitation, myelin basic protein, cyclic nucleotide phosphodiesterase,proteolipid protein, Gtx, and Sox10. Use of an oligodendrocyte-specificor preferred promoter can increase the specificity achieved by thechimeric AAV vector by further limiting expression of the heterologousnucleic acid to oligodendrocytes. Other inducible promoter/enhancerelements include hormone-inducible and metal-inducible elements.Exemplary inducible promoters/enhancer elements include, but are notlimited to, a Tet on/off element, a RU486-inducible promoter, anecdysone-inducible promoter, a rapamycin-inducible promoter, and ametallothionein promoter.

In embodiments wherein the heterologous nucleic acid sequence(s) istranscribed and then translated in the target cells, specific initiationsignals are generally employed for efficient translation of insertedprotein coding sequences. These exogenous translational controlsequences, which may include the ATG initiation codon and adjacentsequences, can be of a variety of origins, both natural and synthetic.

The invention also provides chimeric AAV particles comprising an AAVcapsid and an AAV genome, wherein the AAV genome “corresponds to” (i.e.,encodes) the AAV capsid. Also provided are collections or libraries ofsuch chimeric AAV particles, wherein the collection or library comprises2 or more, 10 or more, 50 or more, 100 or more, 1000 or more, 10⁴ ormore, 10⁵ or more, or 10⁶ or more distinct sequences.

The present invention further encompasses “empty” capsid particles(I.e., in the absence of a vector genome) comprising, consisting of, orconsisting essentially of the chimeric AAV capsid proteins of theinvention. The chimeric AAV capsids of the invention can be used as“capsid vehicles,” as has been described in U.S. Pat. No. 5,863,541.Molecules that can be covalently linked, bound to or packaged by thevirus capsids and transferred into a cell include DNA, RNA, a lipid, acarbohydrate, a polypeptide, a small organic molecule, or combinationsof the same. Further, molecules can be associated with (e.g., “tetheredto”) the outside of the virus capsid for transfer of the molecules intohost target cells. In one embodiment of the invention the molecule iscovalently linked (i.e., conjugated or chemically coupled) to the capsidproteins. Methods of covalently linking molecules are known by thoseskilled in the art.

The virus capsids of the invention also find use in raising antibodiesagainst the novel capsid structures. As a further alternative, anexogenous amino acid sequence may be inserted into the virus capsid forantigen presentation to a cell, e.g., for administration to a subject toproduce an immune response to the exogenous amino acid sequence.

The invention also provides nucleic acids (e.g., isolated nucleic acids)encoding the chimeric virus capsids and chimeric capsid proteins of theinvention. Further provided are vectors comprising the nucleic acids,and cells (in vivo or in culture) comprising the nucleic acids and/orvectors of the invention. Such nucleic acids, vectors and cells can beused, for example, as reagents (e.g., helper constructs or packagingcells) for the production of virus vectors as described herein.

In exemplary embodiments, the invention provides nucleic acid sequencesencoding the AAV capsid of SEQ ID NOS:2-4 or at least 90% identical tothe nucleotide sequence of SEQ ID NO:1. The invention also providesnucleic acids encoding the AAV capsid variants, capsid protein variantsand fusion proteins as described above. In particular embodiments, thenucleic acid hybridizes to the complement of the nucleic acid sequencesspecifically disclosed herein under standard conditions as known bythose skilled in the art and encodes a variant capsid and/or capsidprotein. Optionally, the variant capsid or capsid protein substantiallyretains at least one property of the capsid and/or capsid or capsidprotein encoded by the nucleic acid sequence of SEQ ID NO:1. Forexample, a virus particle comprising the variant capsid or variantcapsid protein can substantially retain the oligodendrocyte tropismprofile of a virus particle comprising a capsid or capsid proteinencoded by a nucleic acid coding sequence of SEQ ID NO:1.

For example, hybridization of such sequences may be carried out underconditions of reduced stringency, medium stringency or even stringentconditions. Exemplary conditions for reduced, medium and stringenthybridization are as follows: (e.g., conditions represented by a washstringency of 35-40% Formamide with 5×Denhardt's solution, 0.5% SDS and1×SSPE at 37° C.; conditions represented by a wash stringency of 40-45%Formamide with 5×Denhardt's solution, 0.5% SDS, and 1×SSPE at 42° C.;and conditions represented by a wash stringency of 50% Formamide with5×Denhardt's solution, 0.5% SDS and 1×SSPE at 42° C., respectively).See, e.g., Sambrook et al., Molecular Cloning A Laboratory Manual (2dEd. 1989) (Cold Spring Harbor Laboratory).

In other embodiments, nucleic acid sequences encoding a variant capsidor capsid protein of the invention have at least about 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher sequence identity with thenucleic acid sequence of SEQ ID NO:1 and optionally encode a variantcapsid or capsid protein that substantially retains at least oneproperty of the capsid or capsid protein encoded by a nucleic acid ofSEQ ID NO:1.

As is known in the art, a number of different programs can be used toidentify whether a nucleic acid or polypeptide has sequence identity toa known sequence. Percent identity as used herein means that a nucleicacid or fragment thereof shares a specified percent identity to anothernucleic acid, when optimally aligned (with appropriate nucleotideinsertions or deletions) with the other nucleic acid (or itscomplementary strand), using BLASTN. To determine percent identitybetween two different nucleic acids, the percent identity is to bedetermined using the BLASTN program “BLAST 2 sequences”. This program isavailable for public use from the National Center for Biotechnologyinformation (NCBI) over the Internet (Altschul et al., (1997) NucleicAcids Res. 25(17):3389-3402). The parameters to be used are whatevercombination of the following yields the highest calculated percentidentity (as calculated below) with the default parameters shown inparentheses: Program—blastn Matrix—0 BLOSUM62 Reward for a match—0 or 1(1) Penalty for a mismatch—0, −1, −2 or −3 (−2) Open gap penalty—0, 1,2, 3, 4 or 5 (5) Extension gap penalty—0 or 1 (1) Gap x_dropoff—0 or 50(50) Expect—10.

Percent identity or similarity when referring to polypeptides, indicatesthat the polypeptide in question exhibits a specified percent identityor similarity when compared with another protein or a portion thereofover the common lengths as determined using BLASTP. This program is alsoavailable for public use from the National Center for BiotechnologyInformation (NCBI) over the Internet (Altschul et al., (1997) NucleicAcids Res. 25(17):3389-3402). Percent identity or similarity forpolypeptides is typically measured using sequence analysis software.See, e.g., the Sequence Analysis Software Package of the GeneticsComputer Group. University of Wisconsin Biotechnology Center, 910University Avenue, Madison, Wis. 53705. Protein analysis softwarematches similar sequences using measures of homology assigned to varioussubstitutions, deletions and other modifications. Conservativesubstitutions typically include substitutions within the followinggroups: glycine, alanine; valine, isoleucine, leucine; aspartic acid,glutamic acid; asparagine, glutamine; serine, threonine; lysine,arginine; and phenylalanine, tyrosine.

In particular embodiments, the nucleic acid can comprise, consistessentially of, or consist of a vector including but not limited to aplasmid, phage, viral vector (e.g., AAV vector, an adenovirus vector, aherpesvirus vector, or a baculovirus vector), bacterial artificialchromosome (BAC), or yeast artificial chromosome (YAC). For example, thenucleic acid can comprise, consist of, or consist essentially of an AAVvector comprising a 5′ and/or 3 terminal repeat (e.g., 5′ and/or 3′ AAVterminal repeat).

In some embodiments, the nucleic acid encoding the chimeric AAV capsidprotein further comprises an AAV rep coding sequence. For example, thenucleic acid can be a helper construct for producing viral stocks.

The invention also provides packaging cells stably comprising a nucleicacid of the invention. For example, the nucleic acid can be stablyincorporated into the genome of the cell or can be stably maintained inan episomal form (e.g., an “EBV based nuclear episome”).

The nucleic acid can be incorporated into a delivery vector, such as aviral delivery vector. To illustrate, the nucleic acid of the inventioncan be packaged in an AAV particle, an adenovirus particle, aherpesvirus particle, a baculovirus particle, or any other suitablevirus particle.

Moreover, the nucleic acid can be operably associated with a promoterelement. Promoter elements are described in more detail herein.

The present invention further provides methods of producing the virusvectors of the invention. In a representative embodiment, the presentinvention provides a method of producing a recombinant virus vector, themethod comprising providing to a cell in vitro, (a) a templatecomprising (i) a heterologous nucleic acid, and (ii) packaging signalsequences sufficient for the encapsidation of the AAV template intovirus particles (e.g., one or more (e.g., two) terminal repeats, such asAAV terminal repeats), and (b) AAV sequences sufficient for replicationand encapsidation of the template into viral particles (e.g., the AAVrep and AAV cap sequences encoding an AAV capsid of the invention). Thetemplate and AAV replication and capsid sequences are provided underconditions such that recombinant virus particles comprising the templatepackaged within the capsid are produced in the cell. The method canfurther comprise the step of collecting the virus particles from thecell. Virus particles may be collected from the medium and/or by lysingthe cells.

In one illustrative embodiment, the invention provides a method ofproducing a rAAV particle comprising an AAV capsid, the methodcomprising: providing a cell in vitro with a nucleic acid encoding achimeric AAV capsid of the invention, an AAV rep coding sequence, an AAVvector genome comprising a heterologous nucleic acid, and helperfunctions for generating a productive AAV infection; and allowingassembly of the AAV particles comprising the AAV capsid andencapsidating the AAV vector genome.

The cell is typically a cell that is permissive for AAV viralreplication. Any suitable cell known in the art may be employed, such asmammalian cells. Also suitable are trans-complementing packaging celllines that provide functions deleted from a replication-defective helpervirus, e.g., 293 cells or other E1a trans-complementing cells.

The AAV replication and capsid sequences may be provided by any methodknown in the art. Current protocols typically express the AAV rep/capgenes on a single plasmid. The AAV replication and packaging sequencesneed not be provided together, although it may be convenient to do so.The AAV rep and/or cap sequences may be provided by any viral ornon-viral vector. For example, the rep/cap sequences may be provided bya hybrid adenovirus or herpesvirus vector (e.g., inserted into the E1aor E3 regions of a deleted adenovirus vector). EBV vectors may also beemployed to express the AAV cap and rep genes. One advantage of thismethod is that EBV vectors are episomal, yet will maintain a high copynumber throughout successive cell divisions (i.e., are stably integratedinto the cell as extra-chromosomal elements, designated as an EBV basednuclear episome.

As a further alternative, the rep/cap sequences may be stably carried(episomal or integrated) within a cell.

Typically, the AAV rep/cap sequences will not be flanked by the AAVpackaging sequences (e.g., AAV ITRs), to prevent rescue and/or packagingof these sequences.

The template (e.g., an rAAV vector genome) can be provided to the cellusing any method known in the art. For example, the template may besupplied by a non-viral (e.g., plasmid) or viral vector. In particularembodiments, the template is supplied by a herpesvirus or adenovirusvector (e.g., inserted into the E1a or E3 regions of a deletedadenovirus). As another illustration, Palombo et al., (1998) J. Virol.72:5025, describe a baculovirus vector carrying a reporter gene flankedby the AAV ITRs. EBV vectors may also be employed to deliver thetemplate, as described above with respect to the rep/cap genes.

In another representative embodiment, the template is provided by areplicating rAAV virus. In still other embodiments, an AAV provirus isstably integrated into the chromosome of the cell.

To obtain maximal virus titers, helper virus functions (e.g., adenovirusor herpesvirus) essential for a productive AAV infection are generallyprovided to the cell. Helper virus sequences necessary for AAVreplication are known in the art. Typically, these sequences areprovided by a helper adenovirus or herpesvirus vector. Alternatively,the adenovirus or herpesvirus sequences can be provided by anothernon-viral or viral vector, e.g., as a non-infectious adenovirusminiplasmid that carries all of the helper genes required for efficientAAV production as described by Ferrari et al., (1997) Nature Med.3:1295, and U.S. Pat. Nos. 6,040,183 and 6,093,570.

Further, the helper virus functions may be provided by a packaging cellwith the helper genes integrated in the chromosome or maintained as astable extrachromosomal element. In representative embodiments, thehelper virus sequences cannot be packaged into AAV virions, e.g., arenot flanked by AAV ITRs.

Those skilled in the art will appreciate that it may be advantageous toprovide the AAV replication and capsid sequences and the helper virussequences (e.g., adenovirus sequences) on a single helper construct.This helper construct may be a non-viral or viral construct, but isoptionally a hybrid adenovirus or hybrid herpesvirus comprising the AAVrep/cap genes.

In one particular embodiment, the AAV rep/cap sequences and theadenovirus helper sequences are supplied by a single adenovirus helpervector. This vector further contains the rAAV template. The AAV rep/capsequences and/or the rAAV template may be inserted into a deleted region(e.g., the E1a or E3 regions) of the adenovirus.

In a further embodiment, the AAV rep/cap sequences and the adenovirushelper sequences are supplied by a single adenovirus helper vector. TherAAV template is provided as a plasmid template.

In another illustrative embodiment, the AAV rep/cap sequences andadenovirus helper sequences are provided by a single adenovirus helpervector, and the rAAV template is integrated into the cell as a provirus.Alternatively, the rAAV template is provided by an EBV vector that ismaintained within the cell as an extrachromosomal element (e.g., as a“EBV based nuclear episome,” see Margolski, (1992) Curr. Top. Microbiol.Immun. 158:67).

In a further exemplary embodiment, the AAV rep/cap sequences andadenovirus helper sequences are provided by a single adenovirus helper.The rAAV template is provided as a separate replicating viral vector.For example, the rAAV template may be provided by a rAAV particle or asecond recombinant adenovirus particle.

According to the foregoing methods, the hybrid adenovirus vectortypically comprises the adenovirus 5′ and 3′ cis sequences sufficientfor adenovirus replication and packaging (i.e., the adenovirus terminalrepeats and PAC sequence). The AAV rep/cap sequences and, if present,the rAAV template are embedded in the adenovirus backbone and areflanked by the 5′ and 3′ cis sequences, so that these sequences may bepackaged into adenovirus capsids. As described above, in representativeembodiments, the adenovirus helper sequences and the AAV rep/capsequences are not flanked by the AAV packaging sequences (e.g., the AAVITRs), so that these sequences are not packaged into the AAV virions.

Herpesvirus may also be used as a helper virus in AAV packaging methods.Hybrid herpesviruses encoding the AAV rep protein(s) may advantageouslyfacilitate for more scalable AAV vector production schemes. A hybridherpes simplex virus type I (HSV-1) vector expressing the AAV-2 rep andcap genes has been described (Conway et al., (1999) Gene Therapy 6:986and WO 00/17377, the disclosures of which are incorporated herein intheir entireties).

As a further alternative, the virus vectors of the invention can beproduced in insect cells using baculovirus vectors to deliver therep/cap genes and rAAV template as described by Urabe et al., (2002)Human Gene Therapy 13:1935-43.

Other methods of producing AAV use stably transformed packaging cells(see, e.g., U.S. Pat. No. 5,658,785).

AAV vector stocks free of contaminating helper virus may be obtained byany method known in the art. For example, AAV and helper virus may bereadily differentiated based on size. AAV may also be separated awayfrom helper virus based on affinity for a heparin substrate (Zolotukhinet al., (1999) Gene Therapy 6:973). In representative embodiments,deleted replication-defective helper viruses are used so that anycontaminating helper virus is not replication competent. As a furtheralternative, an adenovirus helper lacking late gene expression may beemployed, as only adenovirus early gene expression is required tomediate packaging of AAV virus. Adenovirus mutants defective for lategene expression are known in the art (e.g., ts100K and ts149 adenovirusmutants).

The inventive packaging methods may be employed to produce high titerstocks of virus particles, in particular embodiments, the virus stockhas a titer of at least about 10⁵ transducing units (tu)/ml, at leastabout 10⁶ tu/ml, at least about 10⁷ tu/ml, at least about 10⁸ tu/ml, atleast about 10⁹ tu/ml, or at least about 10¹⁰ tu/ml.

The novel capsid protein and capsid structures find use in raisingantibodies, for example, for diagnostic or therapeutic uses or as aresearch reagent. Thus, the invention also provides antibodies againstthe novel capsid proteins and capsids of the invention.

The term “antibody” or “antibodies” as used herein refers to all typesof immunoglobulins, including IgG, IgM, IgA, IgD, and IgE. The antibodycan be monoclonal or polyclonal and can be of any species of origin,including (for example) mouse, rat, rabbit, horse, goat, sheep or human,or can be a chimeric antibody. See, e.g., Walker et al., Mol. Immunol.26, 403-11 (1989), The antibodies can be recombinant monoclonalantibodies, for example, produced according to the methods disclosed inU.S. Pat. No. 4,474,893 or U.S. Pat. No. 4,816,567. The antibodies canalso be chemically constructed, for example, according to the methoddisclosed in U.S. Pat. No. 4,676,980.

Antibody fragments included within the scope of the present inventioninclude, for example, Fab, F(ab′)2, and Fe fragments, and thecorresponding fragments obtained from antibodies other than IgG. Suchfragments can be produced by known techniques. For example, F(ab′)2fragments can be produced by pepsin digestion of the antibody molecule,and Fab fragments can be generated by reducing the disulfide bridges ofthe F(ab′)2 fragments. Alternatively, Fab expression libraries can beconstructed to allow rapid and easy identification of monoclonal Fabfragments with the desired specificity (Huse et al., (1989) Science 254,1275-1281).

Polyclonal antibodies can be produced by immunizing a suitable animal(e.g., rabbit, goat, etc.) with an antigen to which a monoclonalantibody to the target binds, collecting immune serum from the animal,and separating the polyclonal antibodies from the immune serum, inaccordance with known procedures.

Monoclonal antibodies can be produced in a hybridoma cell line accordingto the technique of Kohler and Milstein, (1975) Nature 265, 495-97. Forexample, a solution containing the appropriate antigen can be injectedinto a mouse and, after a sufficient time, the mouse sacrificed andspleen cells obtained. The spleen cells are then immortalized by fusingthem with myeloma cells or with lymphoma cells, typically in thepresence of polyethylene glycol, to produce hybridoma cells. Thehybridoma cells are then grown in a suitable medium and the supernatantscreened for monoclonal antibodies having the desired specificity.Monoclonal Fab fragments can be produced in E. coli by recombinanttechniques known to those skilled in the art. See, e.g., W. Huse, (1989)Science 246, 1275-81.

Antibodies specific to a target polypeptide can also be obtained byphage display techniques known in the art.

Various immunoassays can be used for screening to identify antibodieshaving the desired specificity, Numerous protocols for competitivebinding or immunoradiometric assays using either polyclonal ormonoclonal antibodies with established specificity are well known in theart. Such immunoassays typically involve the measurement of complexformation between an antigen and its specific antibody (e.g.,antigen/antibody complex formation). A two-site, monoclonal-basedimmunoassay utilizing monoclonal antibodies reactive to twonon-interfering epitopes can be used as well as a competitive bindingassay.

Antibodies can be conjugated to a solid support (e.g., beads, plates,slides or wells formed from materials such as latex or polystyrene) inaccordance with known techniques. Antibodies can likewise be directly orindirectly conjugated to detectable groups such as radiolabels (e.g.,³⁵S, ¹²⁵I, ¹³¹I), enzyme labels (e.g., horseradish peroxidase, alkalinephosphatase), and fluorescence labels (e.g., fluorescein) in accordancewith known techniques. Determination of the formation of anantibody/antigen complex in the methods of this invention can be bydetection of, for example, precipitation, agglutination, flocculation,radioactivity, color development or change, fluorescence, luminescence,etc., as is well known in the art.

III. Methods of Using Chimeric AAV Capsids

The present invention also relates to methods for deliveringheterologous nucleotide sequences into oligodendrocytes. The virusvectors of the invention may be employed, e.g., to deliver a nucleotidesequence of interest to an oligodendrocyte in vitro, e.g., to produce apolypeptide or nucleic acid in vitro or for ex vivo gene therapy. Thevectors are additionally useful in a method of delivering a nucleotidesequence to a subject in need thereof, e.g., to express a therapeutic orimmunogenic polypeptide or nucleic acid. In this manner, the polypeptideor nucleic acid may thus be produced in vivo in the subject. The subjectmay be in need of the polypeptide or nucleic acid because the subjecthas a deficiency of the polypeptide, or because the production of thepolypeptide or nucleic acid in the subject may impart some therapeuticeffect, as a method of treatment or otherwise, and as explained furtherbelow.

In particular embodiments, the vectors are useful to express apolypeptide or nucleic acid that provides a beneficial effect tooligodendrocytes, e.g., to promote growth and/or differentiation ofoligodendrocytes. The ability to target vectors to oligodendrocytes maybe particularly useful to treat diseases or disorders involvingoligodendrocyte dysfunction and/or demyelination of neurons. In otherembodiments, the vectors are useful to express a polypeptide or nucleicacid that provides a beneficial effect to cells near theoligodendrocytes (e.g., neurons).

Thus, one aspect of the invention relates to a method of delivering anucleic acid of interest to an oligodendrocyte, the method comprisingcontacting the oligodendrocyte with the AAV particle of the invention.

In another aspect, the invention relates to a method of delivering anucleic acid of interest to an oligodendrocyte in a mammalian subject,the method comprising administering an effective amount of the AAVparticle or pharmaceutical formulation of the invention to a mammaliansubject.

A further aspect of the invention relates to a method of treating adisorder associated with oligodendrocyte dysfunction in a subject inneed thereof, the method comprising administering a therapeuticallyeffective amount of the AAV particle of the invention to the subject. Inone embodiment, the disorder associated with oligodendrocyte dysfunctionis a demyelinating disease. In one embodiment, the disorder associatedwith oligodendrocyte dysfunction is multiple sclerosis,Pelizaeus-Merzbacher disease, Krabbe's disease, metachromaticleukodystrophy, adrenoleukodystrophy, Canavan disease, Alexanderdisease, orthochromatic leukodystrophy, Zellweger disease, 18q-syndrome,cerebral palsy, spinal cord injury, traumatic brain injury, stroke,phenylketonuria, or viral infection, or any other disorder known orlater found to be associated with oligodendrocyte dysfunction. Inanother embodiment, the methods of the invention are used to treat adisorder that is not directly associated with oligodendrocytedysfunction but would benefit by expression of a heterologouspolypeptide or nucleic acid in oligodendrocytes in addition to orinstead of expression in neurons, astrocytes, or other CNS cell types.Examples include, without limitation, neurodegenerative disorders suchas Alzheimer's disease, Parkinson's disease, and Huntington's disease,CNS tumors, and other CNS disorders.

CNS disorders include but are not limited to disorders of thinking andcognition such as schizophrenia and delirium; amnestic disorders;disorders of mood, such as affective disorders and anxiety disorders(including post-traumatic stress disorder, separation anxiety disorder,selective mutism, reactive attachment disorder, stereotypic movementdisorder, panic disorders, agoraphobia, specific phobias, social phobia,obsessive-compulsive disorder, acute stress disorder, generalizedanxiety disorder, substance-induced anxiety disorder and/or anxietydisorder not otherwise specified); disorders of social behavior;disorders of learning and memory, such as learning disorders (e.g.,dyslexia); motor skills disorders; communication disorders (e.g.,stuttering); pervasive developmental disorders (e.g., autistic disorder,Rett's disorder, childhood disintegrative disorder, Asperger's disorder,and/or pervasive developmental disorder not otherwise specified) anddementia. Accordingly, the term “central nervous system disorder”encompasses the disorders listed above as well as depressive disorders(including major depressive disorder, dysthmyic disorder, depressivedisorder not otherwise specified, postpartum depression); seasonalaffective disorder, mania; bipolar disorders (including bipolar Idisorder, bipolar II disorder, cyclothymic disorder, bipolar disordernot otherwise specified); attention-deficit and disruptive behaviordisorders (including attention deficit disorder with hyperactivitydisorder, conduct disorder, oppositional defiant disorder and/ordisruptive behavior disorder not otherwise specified); drugaddiction/substance abuse (including abuse of opiates, amphetamines,alcohol, hallucinogens, cannabis, inhalants, phencyclidine, sedatives,hypnotics, anxyolytics and/or cocaine); alcohol-induced disorders;amphetamine-induced disorders; caffeine-induced disorders;cannabis-induced disorders; cocaine-induced disorders;hallucinogen-induced disorders; inhalant-induced disorders;nicotine-induced disorders; opioid-induced disorders;phencyclidine-induced disorders; sedative, hypnotic oranxyolytic-induced disorders; agitation; apathy; psychoses;irritability; disinhibition; schizophreniform disorder; schizoaffectivedisorder; delusional disorder; brief psychotic disorder, sharedpsychotic disorder, substance-induced psychotic disorder; psychoticdisorder not otherwise specified; unipolar disorders, mood disorders(e.g., mood disorder with psychotic features); somatoform disorders;factitious disorders; disassociative disorders; mental retardation;feeding and eating disorders of infancy or early childhood; eatingdisorders such as anorexia nervosa, bulimnia nervosa and/or eatingdisorder not otherwise specified; sleeping disorders (e.g., dyssomniassuch as primary insomnia, primary hypersomnia, narcolepsy,breathing-related sleep disorder and circadian rhythm sleep disorderand/or parasomnias); impulse control disorders (e.g., kleptomania,pyromania, trichotillomania, pathological gambling and/or intermittentexplosive disorder); adjustment disorders; personality disorders (e.g.,paranoid personality disorder, schizoid personality disorder,schizotypal personality disorder, antisocial personality disorder,borderline personality disorder, histrionic personality disorder,narcissistic personality disorder, avoidant personality disorder,dependent personality disorder and/or obsessive-compulsive personalitydisorder); Tic disorders (e.g., Tourette's disorder, chronic motor orvocal tic disorder, transient tic disorder and/or tic disorder nototherwise specified); elimination disorders; and any combination of theforegoing as well as any other disorder or group of disorders describedin the Diagnostic and Statistical Manual of Mental Disorders—FourthEdition (DSM-IV; the American Psychiatric Association, Washington D.C.,1994). “Central Nervous System disorders” also include other conditionsthat implicate the CNS including but not limited to neurodegenerativedisorders such as Alzheimer's disease, involuntary movement disorderssuch as Parkinson's disease, Huntington's disease, amyotrophic lateralsclerosis (ALS), and the like. Other CNS disorders include withoutlimitation epilepsy, multiple sclerosis, neurogenic pain, psychogenicpain, and migraines. In other embodiments, the CNS disorder encompassesany subset of the foregoing diseases or excludes any one or more of theforegoing conditions. In particular embodiments, the term “centralnervous system disorder” does not encompass benign and/or malignanttumors of the CNS.

In another aspect of the invention, the chimeric AAV capsid and vectorsof the invention are fully- or nearly fully-detargeted vectors that canbe further modified to a desirable tropic profile for targeting of oneor more peripheral organs or tissues as discussed below. In this aspect,the present invention also relates to methods for deliveringheterologous nucleotide sequences into a broad range of cells, includingdividing and non-dividing cells. The virus vectors of the invention maybe employed to deliver a nucleotide sequence of interest to a cell invitro, e.g., to produce a polypeptide in vitro or for ex vivo genetherapy. The vectors are additionally useful in a method of delivering anucleotide sequence to a subject in need thereof, e.g., to express atherapeutic or immunogenic polypeptide or nucleic acid. In this manner,the polypeptide or nucleic acid may thus be produced in vivo in thesubject. The subject may be in need of the polypeptide or nucleic acidbecause the subject has a deficiency of the polypeptide, or because theproduction of the polypeptide or nucleic acid in the subject may impartsome therapeutic effect, as a method of treatment or otherwise, and asexplained further below.

In general, the virus vectors of the invention may be employed todeliver any foreign nucleic acid with a biological effect to treat orameliorate the symptoms associated with any disorder related to geneexpression. Further, the invention can be used to treat any diseasestate for which it is beneficial to deliver a therapeutic polypeptide.Illustrative disease states include, but are not limited to; cysticfibrosis (cystic fibrosis transmembrane regulator protein) and otherdiseases of the lung, hemophilia A (Factor VIII), hemophilia B (FactorIX), thalassemia (β-globin), anemia (erythropoietin) and other blooddisorders, Alzheimer's disease (GDF; neprilysin), multiple sclerosis(β-interferon), Parkinson's disease (glial-cell line derivedneurotrophic factor [GDNF]), Huntington's disease (inhibitory RNAincluding without limitation RNAi such as siRNA or shRNA, antisense RNAor microRNA to remove repeats), amyotrophic lateral sclerosis, epilepsy(galanin, neurotrophic factors), and other neurological disorders,cancer (endostatin, angiostatin, TRAIL, FAS-ligand, cytokines includinginterferons; inhibitory RNA including without limitation RNAi (such assiRNA or shRNA), antisense RNA and microRNA including inhibitory RNAagainst VEGF, the multiple drug resistance gene product or a cancerimmunogen), diabetes mellitus (insulin, PGC-α1, GLP-1, myostatinpro-peptide, glucose transporter 4), muscular dystrophies includingDuchenne and Becker (e.g., dystrophin, mini-dystrophin,micro-dystrophin, insulin-like growth factor I, a sarcoglycan [e.g., α,β, γ], Inhibitory RNA [e.g., RNAi, antisense RNA or microRNA] againstmyostatin or myostatin propeptide, laminin-alpha2, Fukutin-relatedprotein, dominant negative myostatin, follistatin, activin type IIsoluble receptor, anti-inflammatory polypeptides such as the Ikappa Bdominant mutant, sarcospan, utrophin, mini-utrophin, inhibitory RNA[e.g., RNAi, antisense RNA or microRNA] against splice junctions in thedystrophin gene to induce exon skipping [see, e.g., WO/2003/095647],inhibitory RNA (e.g., RNAi, antisense RNA or micro RNA] against U7shRNAs to induce exon skipping [see, e.g., WO/2006/021724], andantibodies or antibody fragments against myostatin or myostatinpropeptide), Gaucher disease (glucocerebrosidase), Hurler's disease(α-L-iduronidase), adenosine deaminase deficiency (adenosine deaminase),glycogen storage diseases (e.g., Fabry disease [α-galactosidase] andPompe disease [lysosomal acid α-glucosidase]) and other metabolicdefects including other lysosomal storage disorders and glycogen storagedisorders, congenital emphysema (al-antitrypsin), Lesch-Nyhan Syndrome(hypoxanthine guanine phosphoribosyl transferase), Niemann-Pick disease(sphingomyelinase), Maple Syrup Urine Disease (branched-chain keto aciddehydrogenase), retinal degenerative diseases (and other diseases of theeye and retina; e.g., PDGF, endostatin and/or angiostatin for maculardegeneration), diseases of solid organs such as brain (includingParkinson's Disease [GDNF], astrocytomas [endostatin, angiostatin and/orRNAi against VEGF], glioblastomas [endostatin, angiostatin and/or RNAiagainst VEGF]), liver (RNAi such as siRNA or shRNA, microRNA orantisense RNA for hepatitis B and/or hepatitis C genes), kidney, heartincluding congestive heart failure or peripheral artery disease (PAD)(e.g., by delivering protein phosphatase inhibitor I [I-1],phospholamban, sarcoplasmic endoreticulum Ca²⁺-ATPase [serca2a], zincfinger proteins that regulate the phospholamban gene, Pim-1, PGC-1α,SOD-1, SOD-2, ECF-SOD, kallikrein, thymosin-β4, hypoxia-inducibletranscription factor [HIF], βarket, β2-adrenergic receptor,β2-adrenergic receptor kinase [βARK], phosphoinositide-3 kinase [PI3kinase], calsarcin, an angiogenic factor, S100A1, parvalbumin, adenylylcyclase type 6, a molecule that effects G-protein coupled receptorkinase type 2 knockdown such as a truncated constitutively activebARKct, an inhibitory RNA [e.g., RNAi, antisense RNA or microRNA]against phospholamban; phospholamban inhibitory or dominant-negativemolecules such as phospholamban S16E, etc.), arthritis (insulin-likegrowth factors), joint disorders (insulin-like growth factors), intimalhyperplasia (e.g., by delivering enos, inos), improve survival of hearttransplants (superoxide dismutase), AIDS (soluble CD4), muscle wasting(insulin-like growth factor 1, myostatin pro-peptide, an anti-apoptoticfactor, follistatin), limb ischemia (VEGF, FGF, PGC-1α, EC-SOD, HIF),kidney deficiency (erythropoietin), anemia (erythropoietin), arthritis(anti-inflammatory factors such as IRAP and TNFα soluble receptor),hepatitis (α-interferon), LDL receptor deficiency (LDL receptor),hyperammonemia (ornithine transcarbamylase), spinal cerebral ataxiasincluding SCA1, SCA2 and SCA3, phenylketonuria (phenylalaninehydroxylase), autoimmune diseases, and the like. The invention canfurther be used following organ transplantation to increase the successof the transplant and/or to reduce the negative side effects of organtransplantation or adjunct therapies (e.g., by administeringimmunosuppressant agents or inhibitory nucleic acids to block cytokineproduction). As another example, bone morphogenic proteins (includingRANKL and/or VEGF) can be administered with a bone allograft, forexample, following a break or surgical removal in a cancer patient.

Exemplary lysosomal storage diseases that can be treated according tothe present invention include without limitation: Hurler's Syndrome (MPSIH), Scheie's Syndrome (MPS IS), and Hurler-Scheie Syndrome (MPS 1H/S)(α-L-iduronidase); Hunter's Syndrome (MPS II) (iduronate sulfatesulfatase); Sanfilippo A Syndrome (MPS IIIA) (Heparan-S-sulfatesulfaminidase), Sanfilippo B Syndrome (MPS IIIB)(N-acetyl-D-glucosaminidase). Sanfilippo C Syndrome (MPS IIIC)(Acetyl-CoA-glucosaminide N-acetyltransferase), Sanfilippo D Syndrome(MPS HID) (N-acetyl-glucosaminine-6-sulfate sulfatase); Morquio Adisease (MPS IVA) (Galactosamine-6-sulfate sulfatase), Morquio B disease(MPS IV B) (β-Galactosidase); Maroteaux-lmay disease (MPS VI)(arylsulfatase B); Sly Syndrome (MPS VII) (s-glucuronidase);hyaluronidase deficiency (MPS IX) (hyaluronidase); sialidosis(mucolipidosis I), mucolipidosis 1 (I-Cell disease)(N-actylglucos-aminyl-1-phosphotransfcrase catalytic subunit),mucolipidosis III (pseudo-Hurler polydystrophy)(N-acetylglucos-aminyl-1-phosphotransferase; type IIIA [catalyticsubunit] and type IIIC [substrate recognition subunit]); GM1gangliosidosis (ganglioside β-galactosidase), GM2 gangliosidosis Type I(Tay-Sachs disease) (β-hexaminidase A), GM2 gangliosidosis type II(Sandhoff's disease) (β-hexosaminidase B); Niemann-Pick disease (Types Aand B) (sphingomyelinase); Gaucher's disease (glucocerebrosidase);Farber's disease (ceraminidase); Fabry's disease (α-galactosidase A);Krabbe's disease (galactosylceramide β-galactosidase); metachromaticleukodystrophy (arylsulfatase A); lysosomal acid lipase deficiencyincluding Wolman's disease (lysosomal acid lipase); Batten disease(juvenile neuronal ceroid lipofuscinosis) (lysosomal trans-membrane CLN3protein) sialidosis (neuranainidase 1); galactosialidosis (Goldberg'ssyndrome) (protective protein/cathepsin A); α-mannosidosis(α-D-mannosidase); β-mannosidosis (β-D-mannosidosis); fucosidosis(α-D-fucosidase); aspartylglucosaminuria (N-Aspartylglucosaminidase);and sialuria (Na phosphate cotransporter).

Exemplary glycogen storage diseases that can be treated according to thepresent invention include, but are not limited to, Type Ia GSD (vonGierke disease) (glucose-6-phosphatase), Type Ib GSD(glucose-6-phosphate translocase), Type Ic GSD (microsomal phosphate orpyrophosphate transporter), Type Id GSD (microsomal glucosetransporter), Type II GSD including Pompe disease or infantile Type IIaGSD (lysosomal acid α-glucosidase) and Type IIb (Danon) (lysosomalmembrane protein-2), Type IIIa and IIIb GSD (Debrancher enzyme;amyloglucosidase and oligoglucanotransferase), Type IV GSD (Andersen'sdisease) (branching enzyme), Type V GSD (McArdle disease) (musclephosphorylase), Type VI GSD (Hers' disease) (liver phosphorylase), TypeVII GSD (Tarui's disease) (phosphofructokinase), GSD Type VIII/IXa(X-linked phosphorylase kinase), GSD Type IXb (Liver and musclephosphorylase kinase), GSD Type IXc (liver phosphorylase kinase), GSDType IXd (muscle phosphorylase kinase), GSD O (glycogen synthase),Fanconi-Bickel syndrome (glucose transporter-2), phosphoglucoisomerasedeficiency, muscle phosphoglycerate kinase deficiency, phosphoglyceratemutase deficiency, fructose 1,6-diphosphatase deficiency,phosphoenolpyruvate carboxykinase deficiency, and lactate debydrogenasedeficiency.

Nucleic acids and polypeptides that can be delivered to cardiac muscleinclude those that are beneficial in the treatment of damaged,degenerated or atrophied cardiac muscle and/or congenital cardiacdefects. For example, angiogenic factors useful for facilitatingvascularization in the treatment of heart disease include but are notlimited to vascular endothelial growth factor (VEGF), VEGF II, VEGF-B,VEGF-C, VEGF-D, VEGF-E, VEGF₁₂₁, VEGF₁₃₈, VEGF₁₄₅, VEGF₁₆₅, VEGF₁₈₉,VEGF₂₀₆, hypoxia inducible factor 1α (HIP 1α), endothelial NO synthase(eNOS), iNOS, VEFGR-1 (Flt1), VEGFR-2 (KDR/Flk1), VEGFR-3 (Flt4),angiogenin, epidermal growth factor (EGF), angiopoietin,platelet-derived growth factor, angiogenic factor, transforming growthfactor-α (TGF-α), transforming growth factor-β (TGF-β), vascularpermeability factor (VPF), tumor necrosis factor alpha (TNF-α),interleukin-3 (IL-3), interleukin-8 (IL-8), platelet-derived endothelialgrowth factor (PD-EGF), granulocyte colony stimulating factor (G-CSF),hepatocyte growth factor (HGF), scatter factor (SF), pleitrophin,proliferin, follistatin, placental growth factor (PIGF), midkine,platelet-derived growth factor-BB-(PDGF), fractalkine, ICAM-1,angiopoietin-1 and -2 (Ang1 and Ang2), Tie-2, neumpilin-1, ICAM-1,chemokines and cytokines that stimulate smooth muscle cell, monocyte, orleukocyte migration, anti-apoptotic peptides and proteins, fibroblastgrowth factors (FGF), FGF-1, FGF-1b, FGF-1c, FGF-2, FGF-2b, FGF-2c,FGF-3, FGF-3b, FGF-3c, FGF-4, FGF-5, FGF-7, FGF-9, acidic FGF, basicFGF, monocyte chemotactic protein-1, granulocyte macrophage-colonystimulating factor, insulin-like growth factor-1 (IGF-1), IGF-2, earlygrowth response factor-1 (EGR-1), ETS-1, human tissue kallikrein (HK),matrix metalloproteinase, chymase, urokinase-type plasminogen activatorand heparinase. (see, e.g., U.S. Patent Application No. 20060287259 andU.S. Patent Application No. 20070059288).

The most common congenital heart disease found in adults is bicuspidaortic valve, whereas atrial septal defect is responsible for 30-40% ofcongenital heart disease seen in adults. The most common congenitalcardiac defect observed in the pediatric population is ventricularseptal defect. Other congenital heart diseases include Eisenmenger'ssyndrome, patent ductus arteriosus, pulmonary stenosis, coarctation ofthe aorta, transposition of the great arteries, tricuspid atresia,univentricular heart, Ebstein's anomaly, and double-outlet rightventricle. A number of studies have identified putative genetic lociassociated with one or more of these congenital heart diseases. Forexample, the putative gene(s) for congenital heart disease associatedwith Down syndrome is 21q22.2-q22.3, between ETS2 and MX1. Similarly,most cases of DiGeorge syndrome result from a deletion of chromosome22q11.2 (the DiGeorge syndrome chromosome region, or DGCR). Severalgenes are lost in this deletion including the putative transcriptionfactor TUPLE1. This deletion is associated with a variety of phenotypes,e.g., Shprintzen syndrome; conotruncal anomaly face (or Takao syndrome);and isolated outflow tract defects of the heart including Tetralogy ofFallot, truncus arteriosus, and interrupted aortic arch. All of theforegoing disorders can be treated according to the present invention.

Other significant diseases of the heart and vascular system are alsobelieved to have a genetic, typically polygenic, etiological component.These diseases include, for example, hypoplastic left heart syndrome,cardiac valvular dysplasia, Pfeiffer cardiocranial syndrome,oculofaciocardiodental syndrome, Kapur-Toriello syndrome, Sonodasyndrome, Ohdo Blepharophimosis syndrome, heart-hand syndrome,Pierre-Robin syndrome, Hirschsprung disease, Kousseff syndrome, Grangeocclusive arterial syndrome, Kearns-Sayre syndrome, Kartagener syndrome,Alagille syndrome, Ritscher-Schinzel syndrome, Ivemark syndrome,Young-Simpson syndrome, hemochromatosis, Holzgreve syndrome, Barthsyndrome, Smith-Lemli-Opitz syndrome, glycogen storage disease,Gaucher-like disease, Fabry disease, Lowry-Maclean syndrome, Rettsyndrome, Opitz syndrome, Marfan syndrome, Miller-Dieker lissencephalysyndrome, mucopolysaccharidosis, Bruada syndrome, humerospinaldysostosis, Phaver syndrome, McDonough syndrome, Marfanoid hypermobilitysyndrome, atransferrinemia, Cornelia de Lange syndrome, Leopardsyndrome, Diamond-Blackfan anemia, Steinfeld syndrome, progeria, andWilliams-Beuren syndrome. All of these disorders can be treatedaccording to the present invention.

Anti-apoptotic factors can be delivered to skeletal muscle, diaphragmmuscle and/or cardiac muscle to treat muscle wasting diseases, limbischemia, cardiac infarction, heart failure, coronary artery diseaseand/or type I or type II diabetes.

Nucleic acids that can be delivered to skeletal muscle include thosethat are beneficial in the treatment of damaged, degenerated and/oratrophied skeletal muscle. The genetic defects that cause musculardystrophy are known for many forms of the disease. These defective geneseither fail to produce a protein product, produce a protein product thatfails to function properly, or produce a dysfunctional protein productthat interferes with the proper function of the cell. The heterologousnucleic acid may encode a therapeutically functional protein or apolynucleotide that inhibits production or activity of a dysfunctionalprotein. Polypeptides that may be expressed from delivered nucleicacids, or inhibited by delivered nucleic acids (e.g., by deliveringRNAi, microRNA or antisense RNA), include without limitation dystrophin,a mini-dystrophin or a micro-dystrophin (Duchene's and Becker MD);dystrophin-associated glycoproteins i-sarcoglycan (limb-girdle MD 2E),δ-sarcoglycan (limb-girdle MD 22F), α-sarcoglycan (limb girdle MI) 2D)and 1-sarcoglycan (limb-girdle MD 2C), utrophin, calpain (autosomalrecessive limb-girdle MD type 2A), caveolin-3 (autosomal-dominantlimb-girdle MD), laminin-alpha2 (merosin-deficient congenital MD),miniagrin (laminin-alpha2 deficient congenital MD), fukutin (Fukuyamatype congenital MD), emerin (Emery-Dreifuss MD), myotilin, lamin A/C,calpain-3, dysferlin, and/or telethonin. Further, the heterologousnucleic acid can encode mir-1, mir-133, mir-206, mir-208 or an antisenseRNA, RNAi (e.g., siRNA or shRNA) or microRNA to induce exon skipping ina defective dystrophin gene.

In particular embodiments, the nucleic acid is delivered to tonguemuscle (e.g., to treat dystrophic tongue). Methods of delivering to thetongue can be by any method known in the art including direct injection,oral administration, topical administration to the tongue, intravenousadministration, intra-articular administration and the like.

The foregoing proteins can also be administered to diaphragm muscle totreat muscular dystrophy.

Alternatively, a gene transfer vector may be administered that encodesany other therapeutic polypeptide.

In particular embodiments, a virus vector according to the presentinvention is used to deliver a nucleic acid of interest as describedherein to skeletal muscle, diaphragm muscle and/or cardiac muscle, forexample, to treat a disorder associated with one or more of thesetissues such as muscular dystrophy, heart disease (including PAD andcongestive heart failure), and the like.

Gene transfer has substantial potential use in understanding andproviding therapy for disease states. There are a number of inheriteddiseases in which defective genes are known and have been cloned. Ingeneral, the above disease states fall into two classes: deficiencystates, usually of enzymes, which are generally inherited in a recessivemanner, and unbalanced states, which may involve regulatory orstructural proteins, and which are typically inherited in a dominantmanner. For deficiency state diseases, gene transfer can be used tobring a normal gene into affected tissues for replacement therapy, aswell as to create animal models for the disease using inhibitory RNAsuch as RNAi (e.g., siRNA or shRNA), microRNA or antisense RNA. Forunbalanced disease states, gene transfer can be used to create a diseasestate in a model system, which can then be used in efforts to counteractthe disease state. Thus, the virus vectors according to the presentinvention permit the treatment of genetic diseases. As used herein, adisease state is treated by partially or wholly remedying the deficiencyor imbalance that causes the disease or makes it more severe. The use ofsite-specific recombination of nucleic sequences to cause mutations orto correct defects is also possible.

The virus vectors according to the present invention may also beemployed to provide an antisense nucleic acid or inhibitory RNA (e.g.,microRNA or RNAi such as a siRNA or shRNA) to a cell in vitro or invivo. Expression of the inhibitory RNA in the target cell diminishesexpression of a particular protein(s) by the cell. Accordingly,inhibitory RNA may be administered to decrease expression of aparticular protein in a subject in need thereof. Inhibitory RNA may alsobe administered to cells in vitro to regulate cell physiology, e.g., tooptimize cell or tissue culture systems.

As a further aspect, the virus vectors of the present invention may beused to produce an immune response in a subject. According to thisembodiment, a virus vector comprising a nucleic acid encoding animmunogen may be administered to a subject, and an active immuneresponse (optionally, a protective immune response) is mounted by thesubject against the immunogen. Immunogens are as described hereinabove.

Alternatively, the virus vector may be administered to a cell ex vivoand the altered cell is administered to the subject. The heterologousnucleic acid is introduced into the cell, and the cell is administeredto the subject, where the heterologous nucleic acid encoding theimmunogen is optionally expressed and induces an immune response in thesubject against the immunogen. In particular embodiments, the cell is anantigen-presenting cell (e.g., a dendritic cell).

An “active immune response” or “active immunity” is characterized by“participation of host tissues and cells after an encounter with theimmunogen. It involves differentiation and proliferation ofimmunocompetent cells in lymphoreticular tissues, which lead tosynthesis of antibody or the development of cell-mediated reactivity, orboth.” Herbert B. Herscowitz, Immunophysiology: Cell Function andCellular Interactions in Antibody Formation, in IMMUNOLOGY: BASICPROCESSES 117 (Joseph A. Bellanti ed., 1985). Alternatively stated, anactive immune response is mounted by the host after exposure toimmunogens by infection or by vaccination. Active immunity can becontrasted with passive immunity, which is acquired through the“transfer of preformed substances (antibody, transfer factor, thymicgraft, interleukin-2) from an actively immunized host to a non-immunehost;” Id

A “protective” immune response or “protective” immunity as used hereinindicates that the immune response confers some benefit to the subjectin that it prevents or reduces the incidence of disease. Alternatively,a protective immune response or protective immunity may be useful in thetreatment of disease, in particular cancer or tumors (e.g., by causingregression of a cancer or tumor and/or by preventing metastasis and/orby preventing growth of metastatic nodules), The protective effects maybe complete or partial, as long as the benefits of the treatmentoutweigh any disadvantages thereof.

The virus vectors of the present invention may also be administered forcancer immunotherapy by administration of a viral vector expressing acancer cell antigen (or an immunologically similar molecule) or anyother immunogen that produces an immune response against a cancer cell.To illustrate, an immune response may be produced against a cancer cellantigen in a subject by administering a viral vector comprising aheterologous nucleotide sequence encoding the cancer cell antigen, forexample to treat a patient with cancer. The virus vector may beadministered to a subject in vivo or by using ex vivo methods, asdescribed herein.

As used herein, the term “cancer” encompasses tumor-forming cancers.Likewise, the term “cancerous tissue” encompasses tumors. A “cancer cellantigen” encompasses tumor antigens.

The term “cancer” has its understood meaning in the art, for example, anuncontrolled growth of tissue that has the potential to spread todistant sites of the body (i.e., metastasize). Exemplary cancersinclude, but are not limited to, leukemia, lymphoma (e.g., Hodgkin andnon-Hodgkin lymphomas), colorectal cancer, renal cancer, liver cancer,breast cancer, lung cancer, prostate cancer, testicular cancer, ovariancancer, uterine cancer, cervical cancer, brain cancer (e.g., gliomas andglioblastoma), bone cancer, sarcoma, melanoma, head and neck cancer,esophageal cancer, thyroid cancer, and the like. In embodiments of theinvention, the invention is practiced to treat and/or preventtumor-forming cancers.

The term “tumor” is also understood in the art, for example, as anabnormal mass of undifferentiated cells within a multicellular organism.Tumors can be malignant or benign. In representative embodiments, themethods disclosed herein are used to prevent and treat malignant tumors.

Cancer cell antigens have been described hereinabove. By the terms“treating cancer” or “treatment of cancer,” it is intended that theseverity of the cancer is reduced or the cancer is prevented or at leastpartially eliminated. For example, in particular contexts, these termsindicate that metastasis of the cancer is prevented or reduced or atleast partially eliminated. In further representative embodiments, theseterms indicate that growth of metastatic nodules (e.g., after surgicalremoval of a primary tumor) is prevented or reduced or at leastpartially eliminated. By the terms “prevention of cancer” or “preventingcancer” it is intended that the methods at least partially eliminate orreduce the incidence or onset of cancer. Alternatively stated, the onsetor progression of cancer in the subject may be slowed, controlled,decreased in likelihood or probability, or delayed.

In particular embodiments, cells may be removed from a subject withcancer and contacted with a virus vector according to the presentinvention. The modified cell is then administered to the subject,whereby an immune response against the cancer cell antigen is elicited.This method is particularly advantageously employed withimmunocompromised subjects that cannot mount a sufficient immuneresponse in vivo (i.e., cannot produce enhancing antibodies insufficient quantities).

It is known in the art that immune responses may be enhanced byimmunomodulatory cytokines (e.g., α-interferon, β-interferon,γ-interferon, ω-interferon, τ-interferon, interleukin-1α,interleukin-1β, interleukin-2, interleukin-3, interleukin-4, interleukin5, interleukin-6, interleukin-7, interleukin-8, interleukin-9,interleukin-10, interleukin-11, interleukin 12, interleukin-13,interleukin-14, interleukin-18, B cell Growth factor, CD40 Ligand, tumornecrosis factor-α, tumor necrosis factor-β, monocyte chemoattractantprotein-1, granulocyte-macrophage colony stimulating factor, andlymphotoxin). Accordingly, immunomodulatory cytokines (e.g., CTLinductive cytokines) may be administered to a subject in conjunctionwith the virus vectors.

Cytokines may be administered by any method known in the art. Exogenouscytokines may be administered to the subject, or alternatively, anucleotide sequence encoding a cytokine may be delivered to the subjectusing a suitable vector, and the cytokine produced in vivo.

The viral vectors are further useful for targeting oligodendrocytes forresearch purposes, e.g., for study of CNS function in vitro or inanimals or for use in creating and/or studying animal models of disease.For example, the vectors can be used to deliver heterologous nucleicacids to oligodendrocytes in animal models of demyelinating diseases.Demyelination can be induced in animals by a variety of means, includingwithout limitation administration of viruses (e.g., Semliki virus,murine hepatitis virus, or Theiler's murine encephalomyelitis virus) andadministration of chemicals (e.g., cuprizone, ethidium bromide, orlysolecithin). In some embodiments, the vector can also be used inanimal models of experimental autoimmune encephalomyelitis. Thiscondition can be induced by, for example, administration of kainite,SIN-1, anti-galactocerebroside, or irradiation. In other embodiments,the vital vector can be used to specifically deliver to oligodendrocytesa toxic agent or an enzyme that produces a toxic agent (e.g., thymidinekinase) in order to kill some or all of the cells.

Further, the virus vectors according to the present invention findfurther use in diagnostic and screening methods, whereby a gene ofinterest is transiently or stably expressed in a cell culture system, oralternatively, a transgenic animal model. The invention can also bepracticed to deliver a nucleic acid for the purposes of proteinproduction, e.g., for laboratory, industrial or commercial purposes.

Recombinant virus vectors according to the present invention find use inboth veterinary and medical applications. Suitable subjects include bothavians and mammals. The term “avian” as used herein includes, but is notlimited to, chickens, ducks, geese, quail, turkeys, pheasant, parrots,parakeets. The term “mammal” as used herein includes, but is not limitedto, humans, primates non-human primates (e.g., monkeys and baboons),cattle, sheep, goats, pigs, horses, cats, dogs, rabbits, rodents (e.g.,rats, mice, hamsters, and the like), etc. Human subjects includeneonates, infants, juveniles, and adults. Optionally, the subject is “inneed of” the methods of the present invention, e.g., because the subjecthas or is believed at risk for a disorder including those describedherein or that would benefit from the delivery of a nucleic acidincluding those described herein. For example, in particularembodiments, the subject has (or has had) or is at risk for ademyelinating disorder or a spinal cord or brain injury. As a furtheroption, the subject can be a laboratory animal and/or an animal model ofdisease.

In particular embodiments, the present invention provides apharmaceutical composition comprising a virus vector of the invention ina pharmaceutically acceptable carrier and, optionally, other medicinalagents, pharmaceutical agents, stabilizing agents, buffers, carriers,adjuvants, diluents, etc. For injection, the carrier will typically be aliquid. For other methods of administration, the carrier may be eithersolid or liquid. For inhalation administration, the carrier will berespirable, and will preferably be in solid or liquid particulate form.

By “pharmaceutically acceptable” it is meant a material that is nottoxic or otherwise undesirable, i.e., the material may be administeredto a subject without causing any undesirable biological effects.

One aspect of the present invention is a method of transferring anucleotide sequence to a cell in vitro. The virus vector may beintroduced to the cells at the appropriate multiplicity of infectionaccording to standard transduction methods appropriate for theparticular target cells. Titers of the virus vector or capsid toadminister can vary, depending upon the target cell type and number, andthe particular virus vector or capsid, and can be determined by those ofskill in the art without undue experimentation. In particularembodiments, at least about 10³ infectious units, more preferably atleast about 10⁵ infectious units are introduced to the cell.

The cell(s) into which the virus vector can be introduced may be of anytype, including but not limited to neural cells (including cells of theperipheral and central nervous systems, in particular, brain cells suchas neurons, oligodendrocytes, glial cells, astrocytes), lung cells,cells of the eye (including retinal cells, retinal pigment epithelium,and corneal cells), epithelial cells (e.g., gut and respiratoryepithelial cells), skeletal muscle cells (including myoblasts, myotubesand myofibers), diaphragm muscle cells, dendritic cells, pancreaticcells (including islet cells), hepatic cells, a cell of thegastrointestinal tract (including smooth muscle cells, epithelialcells), heart cells (including cardiomyocytes), bone cells (e.g., bonemarrow stem cells), hematopoietic stem cells, spleen cells,keratinocytes, fibroblasts, endothelial cells, prostate cells, jointcells (including, e.g., cartilage, meniscus, synovium and bone marrow),germ cells, and the like. Alternatively, the cell may be any progenitorcell. As a further alternative, the cell can be a stem cell (e.g.,neural stem cell, liver stem cell). As still a further alternative, thecell may be a cancer or tumor cell (cancers and tumors are describedabove). Moreover, the cells can be from any species of origin, asindicated above.

The virus vectors may be introduced to cells in vitro for the purpose ofadministering the modified cell to a subject. In particular embodiments,the cells have been removed from a subject, the virus vector isintroduced therein, and the cells are then replaced back into thesubject. Methods of removing cells from subject for treatment ex vivo,followed by introduction back into the subject are known in the art(see, e.g., U.S. Pat. No. 5,399,346). Alternatively, the recombinantvirus vector is introduced into cells from another subject, intocultured cells, or into cells from any other suitable source, and thecells are administered to a subject in need thereof.

Suitable cells for ex vivo gene therapy are as described above. Dosagesof the cells to administer to a subject will vary upon the age,condition and species of the subject, the type of cell, the nucleic acidbeing expressed by the cell, the mode of administration, and the like.Typically, at least about 10² to about 10⁸ or about 10³ to about 10⁶cells will be administered per dose in a pharmaceutically acceptablecarrier. In particular embodiments, the cells transduced with the virusvector are administered to the subject in an effective amount incombination with a pharmaceutical carrier.

In some embodiments, cells that have been transduced with the virusvector may be administered to elicit an immunogenic response against thedelivered polypeptide (e.g., expressed as a transgene or in the capsid).Typically, a quantity of cells expressing an effective amount of thepolypeptide in combination with a pharmaceutically acceptable carrier isadministered. Optionally, the dosage is sufficient to produce aprotective immune response (as defined above). The degree of protectionconferred need not be complete or permanent, as long as the benefits ofadministering the immunogenic polypeptide outweigh any disadvantagesthereof.

A further aspect of the invention is a method of administering the virusvectors or capsids of the invention to subjects. In particularembodiments, the method comprises a method of delivering a nucleic acidof interest to an animal subject, the method comprising: administeringan effective amount of a virus vector according to the invention to ananimal subject. Administration of the virus vectors of the presentinvention to a human subject or an animal in need thereof can be by anymeans known in the art. Optionally, the virus vector is delivered in aneffective dose in a pharmaceutically acceptable carrier.

The virus vectors of the invention can further be administered to asubject to elicit an immunogenic response (e.g., as a vaccine).Typically, vaccines of the present invention comprise an effectiveamount of virus in combination with a pharmaceutically acceptablecarrier. Optionally, the dosage is sufficient to produce a protectiveimmune response (as defined above). The degree of protection conferredneed not be complete or permanent, as long as the benefits ofadministering the immunogenic polypeptide outweigh any disadvantagesthereof. Subjects and immunogens are as described above.

Dosages of the virus vectors to be administered to a subject will dependupon the mode of administration, the disease or condition to be treated,the individual subject's condition, the particular virus vector, and thenucleic acid to be delivered, and can be determined in a routine manner.Exemplary doses for achieving therapeutic effects are virus titers of atleast about 10⁵, 10⁶, 10⁷, 10⁸, 10⁹, 10¹⁰, 10¹¹, 10¹², 10³, 10¹⁴, 10¹⁵transducing units or more, preferably about 10⁷ or 10⁸, 10⁹, 10 ¹⁰,10¹¹, 10¹², 10¹³ or 10¹⁴ transducing units, yet more preferably about10¹² transducing units.

In particular embodiments, more than one administration (e.g., two,three, four or more administrations) may be employed to achieve thedesired level of gene expression over a period of various intervals,e.g., daily, weekly, monthly, yearly, etc.

Exemplary modes of administration include oral, rectal, transmucosal,topical, intranasal, inhalation (e.g., via an aerosol), buccal (e.g.,sublingual), vaginal, intrathecal, intraocular, transdermal, in utero(or in ovo), parenteral (e.g., intravenous, subcutaneous, intradermal,intramuscular [including administration to skeletal, diaphragm and/orcardiac muscle], intradermal, intrapleural, intracerebral, andintraarticular), topical (e.g., to both skin and mucosal surfaces,including airway surfaces, and transdermal administration),intro-lymphatic, and the like, as well as direct tissue or organinjection (e.g., to liver, skeletal muscle, cardiac muscle, diaphragmmuscle or brain). Administration can also be to a tumor (e.g., in or anear a tumor or a lymph node). The most suitable route in any given casewill depend on the nature and severity of the condition being treatedand on the nature of the particular vector that is being used.

In some embodiments, the viral vector is administered directly to theCNS, e.g., the brain or the spinal cord. Direct administration canresult in high specificity of transduction of oligodendrocytes, e.g.,wherein at least 80%, 85%, 90%, 95% or more of the transduced cells areoligodendrocytes. Any method known in the art to administer vectorsdirectly to the CNS can be used. The vector may be introduced into thespinal cord, brainstem (medulla oblongata, pons), midbrain(hypothalamus, thalamus, epithalamus, pituitary gland, substantia nigra,pineal gland), cerebellum, telencephalon (corpus striatum, cerebrumincluding the occipital, temporal, parietal and frontal lobes, cortex,basal ganglia, hippocampus and amygdala), limbic system, neocortex,corpus striatum, cerebrum, and inferior colliculus. The vector may alsobe administered to different regions of the eye such as the retina,cornea or optic nerve. The vector may be delivered into thecerebrospinal fluid (e.g., by lumbar puncture) for more disperseadministration of the vector.

The delivery vector may be administered to the desired region(s) of theCNS by any route known in the art, including but not limited to,intrathecal, intracerebral, intraventricular, intranasal, intra-aural,intra-ocular (e.g., intra-vitreous, sub-retinal, anterior chamber) andperi-ocular (e.g., sub-Tenon's region) delivery or any combinationthereof.

Typically, the viral vector will be administered in a liquid formulationby direct injection (e.g., stereotactic injection) to the desired regionor compartment in the CNS. In some embodiments, the vector can bedelivered via a reservoir and/or pump. In other embodiments, the vectormay be provided by topical application to the desired region or byintra-nasal administration of an aerosol formulation. Administration tothe eye or into the ear, may be by topical application of liquiddroplets. As a further alternative, the vector may be administered as asolid, slow-release formulation. Controlled release of parvovirus andAAV vectors is described by international patent publication WO01/91803.

In some embodiments where the subject has a compromised blood-brainbarrier (BBB), the viral vector can be delivered systemically (e.g.,intravenously) to the subject, wherein the vector transducesoligodendrocytes in the area of (e.g., bordering) the BBB compromise. Incertain embodiments, the vector transduces cells in the compromised areabut not cells in uncompromised areas. Thus, one aspect of the inventionrelates to a method of delivering a nucleic acid of interest to an areaof the CNS bordering a compromised blood brain barrier area in amammalian subject, the method comprising intravenously administering aneffective amount of the AAV particle of the invention.

In some embodiments, the compromise in the BBB is due to a disease ordisorder. Examples include, without limitation, neurodegenerativediseases such as Alzheimer's, Parkinson's disease, disease, amyotrophiclateral sclerosis, and multiple sclerosis, epilepsy, CNS tumors, orcerebral infarcts. In other embodiments, the BBB compromise can be aninduced disruption, e.g., to promote delivery of agents to the CNS.Temporary BBB compromises can be induced by, for example, toxicchemicals (such as metrazol, VP-16, cisplatin, hydroxyurea,fluorouracil, and etoposide), osmotic agents (such as mannitol andarabinose), biological agents (such as retinoic acid, phorbol myristateacetate, leukotriene C4, bradykinin, histamine, RMP-7, andalkylglycerols), or irradiation (such as ultrasound or electromagneticradiation).

Administration to skeletal muscle according to the present inventionincludes but is not limited to administration to skeletal muscles in thelimbs (e.g., upper arm, lower arm, upper leg, and/or lower leg), back,neck, head (e.g., tongue), thorax, abdomen, pelvis/perineum, and/ordigits. Suitable skeletal muscle tissues include but are not limited toabductor digiti minimi (in the hand), abductor digiti minimi (in thefoot), abductor hallucis, abductor ossis metatarsi quinti, abductorpollicis brevis, abductor pollicis longus, adductor brevis, adductorhallucis, adductor longus, adductor magnus, adductor pollicis, anconeus,anterior scalene, articularis genus, biceps brachii, biceps femoris,brachialis, brachioradialis, buccinator, coracobrachialis, corrugatorsupercilii, deltoid, depressor anguli oris, depressor labii inferioris,digastric, dorsal interossei (in the hand), dorsal interossei (in thefoot), extensor carpi radialis brevis, extensor carpi radialis longus,extensor carpi ulnaris, extensor digiti minimi, extensor digitorum,extensor digitorum brevis, extensor digitorum longus, extensor hallucisbrevis, extensor hallucis longus, extensor indicis, extensor pollicisbrevis, extensor pollicis longus, flexor carpi radialis, flexor carpiulnaris, flexor digiti minimi brevis (in the hand), flexor digiti minimibrevis (in the foot), flexor digitorum brevis, flexor digitorum longus,flexor digitorum profundus, flexor digitorum superficialis, flexorhallucis brevis, flexor hallucis longus, flexor pollicis brevis, flexorpollicis longus, frontalis, gastrocnemius, geniohyoid, gluteus maximus,gluteus medius, gluteus minimus, gracilis, iliocostalis cervicis,iliocostalis lumborum, iliocostalis thoracis, illiacus, inferiorgemellus, inferior oblique, inferior rectus, infraspinatus,interspinalis, intertransversi, lateral pterygoid, lateral rectus,latissimus dorsi, levator anguli oris, levator labii superioris, levatorlabii superioris alaeque nasi, levator palpebrae superioris, levatorscapulae, long rotators, longissimus capitis, longissimus cervicis,longissimus thoracis, longus capitis, longus colli, lumbricals (in thehand), lumbricals (in the foot), masseter, medial pterygoid, medialrectus, middle scalene, muitifidus, mylohyoid, obliquus capitisinferior, obliquus capitis superior, obturator externus, obturatorinternus, occipitalis, omohyoid, opponens digiti minimi, opponenspollicis, orbicularis oculi, orbicularis oris, palmar interossei,palmaris brevis, palmaris longus, pectineus, pectoralis major,pectoralis minor, peroneus brevis, peroneus longus, peroneus tertius,piriformis, plantar interossei, plantaris, platysma, popliteus,posterior scalene, pronator quadratus, pronator teres, psoas major,quadratus femoris, quadratus plantae, rectus capitis anterior, rectuscapitis lateralis, rectus capitis posterior major, rectus capitisposterior minor, rectus femoris, rhomboid major, rhomboid minor,risorius, sartorius, scalenus minimus, semimembranosus, semispinaliscapitis, semispinalis cervicis, semispinalis thoracis, semitendinosus,serratus anterior, short rotators, soleus, spinalis capitis, spinaliscervicis, spinalis thoracis, splenius capitis, splenius cervicis,sternocleidomastoid, sternohyoid, sternothyroid, stylohyoid, subclavius,subscapularis, superior gemellus, superior oblique, superior rectus,supinator, supraspinatus, temporalis, tensor fascia lata, teres major,teres minor, thoracis, thyrohyoid, tibialis anterior, tibialisposterior, trapezius, triceps brachii, vastus intennedius, vastuslateralis, vastus medialis, zygomaticus major, and zygomaticus minor andany other suitable skeletal muscle as known in the art.

The virus vector can be delivered to skeletal muscle by any suitablemethod including without limitation intravenous administration,intra-arterial administration, intraperitoneal administration, isolatedlimb perfusion (of leg and/or arm; see, e.g. Arruda et al., (2005) Blood105:3458-3464), and/or direct intramuscular injection.

Administration to cardiac muscle includes without limitationadministration to the left atrium, right atrium, left ventricle, rightventricle and/or septum. The virus vector can be delivered to cardiacmuscle by any method known in the art including, e.g., intravenousadministration, intra-arterial administration such as intra-aorticadministration, direct cardiac injection (e.g., into left atrium, rightatrium, left ventricle, right ventricle), and/or coronary arteryperfusion.

Administration to diaphragm muscle can be by any suitable methodincluding intravenous administration, intra-arterial administration,and/or intra-peritoneal administration.

Delivery to any of these tissues can also be achieved by delivering adepot comprising the virus vector, which can be implanted into theskeletal, cardiac and/or diaphragm muscle tissue or the tissue can becontacted with a film or other matrix comprising the virus vector.Examples of such implantable matrices or substrates are described inU.S. Pat. No. 7,201,898.

In particular embodiments, a virus vector according to the presentinvention is administered to skeletal muscle, diaphragm muscle and/orcardiac muscle (e.g., to treat muscular dystrophy or heart disease [forexample, PAD or congestive heart failure]).

The invention can be used to treat disorders of skeletal, cardiac and/ordiaphragm muscle. Alternatively, the invention can be practiced todeliver a nucleic acid to skeletal, cardiac and/or diaphragm muscle,which is used as a platform for production of a protein product (e.g.,an enzyme) or non-translated RNA (e.g., RNAi, microRNA, antisense RNA)that normally circulates in the blood or for systemic delivery to othertissues to treat a disorder (e.g., a metabolic disorder, such asdiabetes (e.g., insulin), hemophilia (e.g., Factor IX or Factor VIII),or a lysosomal storage disorder (such as Gaucher's disease[glucocerebrosidase], Pompe disease [lysosomal acid α-glucosidase] orFabry disease [α-galactosidase A]) or a glycogen storage disorder (suchas Pompe disease [lysosomal acid α glucosidase]). Other suitableproteins for treating metabolic disorders are described above.

In a representative embodiment, the invention provides a method oftreating muscular dystrophy in a subject in need thereof, the methodcomprising: administering an effective amount of a virus vector of theinvention to a mammalian subject, wherein the virus vector comprises aheterologous nucleic acid effective to treat muscular dystrophy. In anexemplary embodiment, the method comprises: administering an effectiveamount of a virus vector of the invention to a mammalian subject,wherein the virus vector comprises a heterologous nucleic acid encodingdystrophin, a mini-dystrophin, a micro-dystrophin, utrophin,mini-utrophin, laminin-α2, mini-agrin, Fukutin-related protein,follistatin, dominant negative myostatin, α-sarcoglycan, β-sarcoglycan,γ-sarcoglycan, δ-sarcoglycan, IGF-1, myostatin pro-peptide, activin typeII soluble receptor, anti-inflammatory polypeptides such as the Ikappa Bdominant mutant, sarcospan, antibodies or antibody fragments againstmyostatin or myostatin propeptide, or an inhibitory RNA (e.g., antisenseRNA, microRNA or RNAi) against myostatin, mir-1, mir-133, mir-206,mir-208 or an inhibitory RNA (e.g., microRNA, RNAi or antisense RNA) toinduce exon skipping in a defective dystrophin gene. In particularembodiments, the virus vector can be administered to skeletal, diaphragmand/or cardiac muscle as described elsewhere herein.

The invention further encompasses a method of treating a metabolicdisorder in a subject in need thereof. In representative embodiments,the method comprises: administering an effective amount of a virusvector of the invention to skeletal muscle of a subject, wherein thevirus vector comprises, a heterologous nucleic acid encoding apolypeptide, wherein the metabolic disorder is a result of a deficiencyand/or defect in the polypeptide. Illustrative metabolic disorders andheterologous nucleic acids encoding polypeptides are described herein.As a further option, the heterologous nucleic acid can encode a secretedprotein.

The invention can also be practiced to produce inhibitory RNA (e.g.,antisense RNA, microRNA or RNAi) for systemic delivery.

The invention also provides a method of treating congenital heartfailure in a subject in need thereof, the method comprisingadministering an effective amount of a virus vector of the invention toa mammalian subject, wherein the virus vector comprises a heterologousnucleic acid effective to treat congenital heart failure. Inrepresentative embodiments, the method comprises administering aneffective amount of a virus vector of the invention to a mammaliansubject, wherein the virus vector comprises a heterologous nucleic acidencoding a sarcoplasmic endoreticulum Ca²⁺-ATPase (SERCA2a), anangiogenic factor, phospholamban, PI3 kinase, calsarcan, a β-adrenergicreceptor kinase (βARK), βARKct, inhibitor 1 of protein phosphatase 1,Pim-1, PGC-1α, SOD-1, SOD-2, EC-SOD, Kallikrein, HIF, thymosin-β4,S100A1, parvalbumin, adenylyl cyclase type 6, a molecule that effectsG-protein coupled receptor kinase type 2 knockdown such as a truncatedconstitutively active bARKet; phospholamban inhibitory ordominant-negative molecules such as phospholamban S16E, mir-1, mir-133,mir-206, mir-208.

Injectables can be prepared in conventional forms, either as liquidsolutions or suspensions, solid forms suitable for solution orsuspension in liquid prior to injection, or as emulsions. Alternatively,one may administer the virus vector in a local rather than systemicmanner, for example, in a depot or sustained-release formulation.Further, the virus vector can be delivered dried to a surgicallyimplantable matrix such as a bone graft substitute, a suture, a stent,and the like (e.g., as described in U.S. Pat. No. 7,201,898).

Pharmaceutical compositions suitable for oral administration can bepresented in discrete units, such as capsules, cachets, lozenges, ortablets, each containing a predetermined amount of the composition ofthis invention; as a powder or granules; as a solution or a suspensionin an aqueous or non-aqueous liquid; or as an oil-in-water orwater-in-oil emulsion. Oral delivery can be performed by complexing avirus vector of the present invention to a carrier capable ofwithstanding degradation by digestive enzymes in the gut of an animal.Examples of such carriers include plastic capsules or tablets, as knownin the art. Such formulations are prepared by any suitable method ofpharmacy, which includes the step of bringing into association thecomposition and a suitable carrier (which may contain one or moreaccessory ingredients as noted above). In general, the pharmaceuticalcomposition according to embodiments of the present invention areprepared by uniformly and intimately admixing the composition with aliquid or finely divided solid carrier, or both, and then, if necessary,shaping the resulting mixture. For example, a tablet can be prepared bycompressing or molding a powder or granules containing the composition,optionally with one or more accessory ingredients. Compressed tabletsare prepared by compressing, in a suitable machine, the composition in afree-flowing form, such as a powder or granules optionally mixed with abinder, lubricant, inert diluent, and/or surface active/dispersingagent(s). Molded tablets are made by molding, in a suitable machine, thepowdered compound moistened with an inert liquid binder.

Pharmaceutical compositions suitable for buccal (sub-lingual)administration include lozenges comprising the composition of thisinvention in a flavored base, usually sucrose and acacia or tragacanth;and pastilles comprising the composition in an inert base such asgelatin and glycerin or sucrose and acacia.

Pharmaceutical compositions suitable for parenteral administration cancomprise sterile aqueous and non-aqueous injection solutions of thecomposition of this invention, which preparations are optionallyisotonic with the blood of the intended recipient. These preparationscan contain anti-oxidants, buffers, bacteriostats and solutes, whichrender the composition isotonic with the blood of the intendedrecipient. Aqueous and non-aqueous sterile suspensions, solutions andemulsions can include suspending agents and thickening agents. Examplesof non-aqueous solvents are propylene glycol, polyethylene glycol,vegetable oils such as olive oil, and injectable organic esters such asethyl oleate. Aqueous carriers include water, alcoholic/aqueoussolutions, emulsions or suspensions, including saline and bufferedmedia. Parenteral vehicles include sodium chloride solution, Ringer'sdextrose, dextrose and sodium chloride, lactated Ringer's, or fixedoils. Intravenous vehicles include fluid and nutrient replenishers,electrolyte replenishers (such as those based on Ringer's dextrose), andthe like. Preservatives and other additives may also be present such as,for example, antimicrobials, anti-oxidants, chelating agents, and inertgases and the like.

The compositions can be presented in unit/dose or multi-dose containers,for example, in sealed ampoules and vials, and can be stored in afreeze-dried (lyophilized) condition requiring only the addition of thesterile liquid carrier, for example, saline or water-for-injectionimmediately prior to use.

Extemporaneous injection solutions and suspensions can be prepared fromsterile powders, granules and tablets of the kind previously described.For example, an injectable, stable, sterile composition of thisinvention in a unit dosage form in a sealed container can be provided.The composition can be provided in the form of a lyophilizate, which canbe reconstituted with a suitable pharmaceutically acceptable carrier toform a liquid composition suitable for injection into a subject. Theunit dosage form can be from about 1 μg to about 10 grams of thecomposition of this invention. When the composition is substantiallywater-insoluble, a sufficient amount of emulsifying agent, which isphysiologically acceptable, can be included in sufficient quantity toemulsify the composition in an aqueous carrier. One such usefulemulsifying agent is phosphatidyl choline.

Pharmaceutical compositions suitable for rectal administration can bepresented as unit dose suppositories. These can be prepared by admixingthe composition with one or more conventional solid carriers, such asfor example, cocoa butter and then shaping the resulting mixture.

Pharmaceutical compositions of this invention suitable for topicalapplication to the skin can take the form of an ointment, cream, lotion,paste, gel, spray, aerosol, and/or oil. Carriers that can be usedinclude, but are not limited to, petroleum jelly, lanoline, polyethyleneglycols, alcohols, transdermal enhancers, and combinations of two ormore thereof. In some embodiments, for example, topical delivery can beperformed by mixing a pharmaceutical composition of the presentinvention with a lipophilic reagent (e.g., DMSO) that is capable ofpassing into the skin.

Pharmaceutical compositions suitable for transdermal administration canbe in the form of discrete patches adapted to remain in intimate contactwith the epidermis of the subject for a prolonged period of time.Compositions suitable for transdermal administration can also bedelivered by iontophoresis (see, for example, Pharm. Res. 3:318 (1986))and typically take the form of an optionally buffered aqueous solutionof the composition of this invention. Suitable formulations can comprisecitrate or bis\tris buffer (pH 6) or ethanol/water and can contain from0.1 to 0.2M active ingredient.

The virus vectors disclosed herein may be administered to the lungs of asubject by any suitable means, for example, by administering an aerosolsuspension of respirable particles comprised of the virus vectors, whichthe subject inhales. The respirable particles may be liquid or solid.Aerosols of liquid particles comprising the virus vectors may beproduced by any suitable means, such as with a pressure-driven aerosolnebulizer or an ultrasonic nebulizer, as is known to those of skill inthe art. See, e.g., U.S. Pat. No. 4,501,729. Aerosols of solid particlescomprising the virus vectors may likewise be produced with any solidparticulate medicament aerosol generator, by techniques known in thepharmaceutical art.

IV. Use of the AAV Capsid to Target Peripheral Tissues

The AAV capsids and vectors of the present invention have beendemonstrated to be fully or nearly fully detargeted for peripheralorgans and tissues (see FIG. 4). This detargeting makes the vectorsideal as a “blank” vector that can be altered to produce the desiredtropic profile, e.g., to target specific organs and tissues and/ordetarget other organs and tissues. Thus, one aspect of the inventionrelates to a method of preparing an AAV capsid having a tropism profileof interest, the method comprising modifying the AAV capsid of thepresent invention to insert an amino acid sequence providing the tropismprofile of interest. In some embodiments, the tropism profile ofinterest is enhanced selectivity for a tissue selected from skeletalmuscle, cardiac muscle, diaphragm, kidney, liver, pancreas, spleen,gastrointestinal tract, lung, joint tissue, tongue, ovary, testis, agerm cell, a cancer cell, or a combination thereof and/or reducedselectivity for a tissue selected from liver, ovary, testis, a germcell, or a combination thereof.

Examples of specific targeting and detargeting sequences are known inthe art. One example is the molecular basis for preferential livertropism, which has been mapped, in the case of AAV2 and AAV6, to acontinuous basic footprint that appears to be involved in theinteraction of either serotype with heparin. Specifically, it haspreviously been demonstrated that a single lysine residue on AAV6 (K531)dictates heparin binding ability and consequently, liver tropism. Incorollary, substitutional mutagenesis of the correspondingglutamate/aspartate residue on other serotypes with a lysine residueconfers heparin binding, possibly by forming a minimum continuous basicfootprint on the capsid surface. Another example is the capsid mutantscomprising alterations in the three-fold axis loop 4 as disclosed inInternational Publication No. WO 2012/093784, incorporated herein byreference in its entirety. These mutants exhibit one or more propertiesincluding (i) reduced transduction of liver, (ii) enhanced movementacross endothelial cells, (iii) systemic transduction; (iv) enhancedtransduction of muscle tissue (e.g., skeletal muscle, cardiac muscleand/or diaphragm muscle), and/or (v) reduced transduction of braintissues (e.g., neurons). Other tropic sequences are described in Li etal., (2012) J. Virol. 86:7752-7759; Pulicherla et al., (2011) Mol. Ther,19:1070-1078; Bowles et al., (2012) Mol. Ther. 20:443-455; Asokan etal., (2012) Mol. Ther. 20:699-708; and Asokan et al., (2010) NatureBiotechnol. 28:79-82; each incorporated by reference in its entirety.

In some embodiments, the AAV capsid of the present invention can bemodified through DNA scrambling and/or directed evolution to identifymodified capsids having the desired tropism profile. Techniques for DNAscrambling and directed evolution of AAV capsids are described inInternational Publication No. WO 2009/137006, incorporated herein byreference in its entirety.

Having described the present invention, the same will be explained ingreater detail in the following examples, which are included herein forillustration purposes only, and which are not intended to be limiting tothe invention.

Example 1 Discovery and Characterization of the BNP61 AAV Clone

A mutant DNA shuffled AAV capsid library was injected intravenously intoa rat model of Parkinson's disease and 3 days later cells weredissociated from the caudate nucleus. Using PCR rescue, a single cloneemerged (BNP61). Additional shuffling and selection yielded the sameclone. As seen in FIG. 1, this clone is a chimera of several AAVserotypes.

Direct infusion of this BNP61 clone into the rat brain produced asurprising cellular transduction pattern. To date, almost all diverseAAV serotypes and chimeras exhibit a >95% tropism for neurons, when geneexpression is driven by a constitutive promoter. In marked contrast,BNP61 exhibited a >95% tropism for oligodendrocytes with no evidence ofastrocyte or microglial transduction and minimal neuronal transduction(FIGS. 2A-2C). FIG. 2A shows GFP positive oligodendrocytes in the ratcaudate 1 week after the infusion of BNP61-CBh-GFP vectors. Note thatthere are no GP positive neurons. FIG. 2B shows a higher magnificationthat reflects clear oligodendrocyte morphology, and FIG. 2C shows thatnone of the GFP positive cells colocalize with the cellular marker forastrocytes, GFAP (red).

Also, in primary oligodendrocyte cultures, BNP61 transduces theoligodendrocytes but not the underlying bed of astrocytes (FIGS. 3A-3B).Mixed glial cultures at day 10 were transduced with BNP61-GFP at a MOIof 100 viral particles and images were taken 72 hr later. FIG. 3A is alight image of the glial culture. FIG. 3B is an image of fluorescent AAVGFP-positive cells taken from the same frame as FIG. 3A. The underlayerbed of astrocytes was not transduced and nearly all GFP-expressing cellsappear morphologically as oligodendrocytes. Arrows indicate near-focusedoligodendrocytes showing processes consistent with oligodendrocyteprogenitor culture morphology. Thus, the BNP61 AAV vector exhibitsproperties that are distinctly different from other AAV vectorscharacterized to date.

In order to assess peripheral biodistribution, mice received intravenousadministration of BNP61-CBh-GFP vectors and subsequently the peripheralorgans were harvested. All mice were injected as adults with 5×10¹⁰ vgexcept as indicated. Neonatal injections were with 2.5×10¹⁰ vg. Adultswere sacrificed 10 days post-injection, and neonates were sacrificed at4 weeks post-injection. * indicates samples not tested. In the legend,the number of animals for each vector is shown in parentheses. Errorbars are S.E.M. As seen in FIG. 4, BNP61 did not accumulate in any ofthe peripheral organs.

Example 2 BNP61 Crosses the Compromised Blood-Brain Barrier

After the first selection round, the BNP61 clone was packaged with GFPand recombinant virus was produced. Then, 2 weeks post-6-OHDA treatmentthe recombinant virus was administered intravenously at a dose of 8×10¹¹vector genomes/kg. One month later, the rats were sacrificed and thebrains sectioned. In these 2 week post treatment rats, substantial geneexpression was found in oligodendrocytes and some neurons within the6-OHDA treated striatum (FIG. 5), while no gene expression was found inthe contralateral striatum, the injector tract in the cortex or distalbrain structures. Note the lack of NeuN co-localization with the manyoligodendrocytes that surround the lone NeuN positive neuron (FIG. 5).Thus, it appears that after intravenous administration this novel AAVclone exhibits the ability to cross the 6-OHDA compromised blood-brainbarrier, but not the intact blood-brain barrier.

Example 3 Generation of Additional Oligodendrocyte Targeted Clones

Using the same AAV DNA capsid shuffling and in vivo directed evolutionprocess described in Example 1, two additional oligodendrocyte-targetedclones were identified. BNP62 and BNP63. As shown in FIG. 1 and similarto BNP61, these clones are chimeras of several AAV serotypes. The aminoacid sequence identity between the three clones is shown in Table 2. Allthree clones are greater than 95% identical to each other at the aminoacid level.

TABLE 2 Sequence identity between clones BNP61 BNP62 BNP63 BNP61 100%96% 97% BNP62 96% 100% 96% BNP63 97% 96% 100%

FIG. 6 shows that the BNP63 clone transduces oligodendrocytes in the ratpiriform cortex. The BNP63 transduced cells (green) do not co-localizewith a cellular marker for astrocytes (GFAP, red). Also, the transducedcells exhibit a clear oligodendrocyte morphology.

The foregoing is illustrative of the present invention, and is not to beconstrued as limiting thereof. The invention is defined by the followingclaims, with equivalents of the claims to be included therein.

1-27. (canceled)
 28. A method of delivering a nucleic acid of interestto an oligodendrocyte, the method comprising contacting theoligodendrocyte with an AAV particle comprising: an AAV vector genomecomprising or encoding the nucleic acid of interest; and an AAV capsidcomprising the amino acid sequence of one of SEQ ID NOS:2-4, wherein 25or fewer amino acids are substituted, deleted, and/or inserted, whereinthe AAV capsid encapsidates the AAV vector genome.
 29. A method ofdelivering a nucleic acid of interest to an oligodendrocyte in amammalian subject, the method comprising: administering an effectiveamount of an AAV particle comprising an AAV vector genome comprising orencoding the nucleic acid of interest; and an AAV capsid comprising theamino acid sequence of one of SEQ ID NOS:2-4, wherein 25 or fewer aminoacids are substituted, deleted, and/or inserted, wherein the AAV capsidencapsidates the AAV vector genome, or a pharmaceutical formulationcomprising the AAV particle to a mammalian subject.
 30. The method ofclaim 29, wherein the mammalian subject is a human subject.
 31. Themethod of claim 29, wherein the AAV particle is delivered to the centralnervous system (CNS).
 32. The method of claim 31, wherein the AAVparticle is delivered directly to the CNS by intrathecal, intracerebral,intraventricular, intranasal, intra-aural, intra-ocular, or peri-oculardelivery, or any combination thereof.
 33. The method of claim 31,wherein the subject has a compromised blood brain barrier and the AAVparticle is delivered to the CNS by intravenous administration.
 34. Amethod of delivering a nucleic acid of interest to an area of the CNSbordering a compromised blood brain barrier area in a mammalian subject,the method comprising: intravenously administering an effective amountof an AAV particle comprising: an AAV vector genome comprising orencoding the nucleic acid of interest; and an AAV capsid comprising theamino acid sequence of one of SEQ ID NOS:2-4, wherein 25 or fewer aminoacids are substituted, deleted, and/or inserted, wherein the AAV capsidencapsidates the AAV vector genome, or a pharmaceutical formulationcomprising the AAV particle to a mammalian subject.
 35. A method oftreating a disorder associated with oligodendrocyte dysfunction in amammalian subject in need thereof, the method comprising administering atherapeutically effective amount of an AAV particle comprising: an AAVvector genome; and an AAV capsid comprising the amino acid sequence ofone of SEQ ID NOS:2-4, wherein 25 or fewer amino acids are substituted,deleted, and/or inserted, wherein the AAV capsid encapsidates the AAVvector genome, or a pharmaceutical formulation comprising the AAVparticle to a mammalian subject.
 36. The method of claim 35, wherein thedisorder associated with oligodendrocyte dysfunction is a demyelinatingdisease.
 37. The method of claim 35, wherein the disorder associatedwith oligodendrocyte dysfunction is multiple sclerosis,Pelizaeus-Merzbacher disease, Krabbe's disease, metachromaticleukodystrophy, adrenoleukodystrophy, Canavan disease, Alexanderdisease, orthochromatic leukodystrophy, Zellweger disease, 18q-syndrome,cerebral palsy, spinal cord injury, traumatic brain injury, stroke,phenylketonuria, or viral infection. 38-39. (canceled)
 40. The method ofclaim 28, wherein the AAV capsid comprises the amino acid sequence ofone of SEQ ID NOS:2-4, wherein 10 or fewer amino acids are substituted,deleted, and/or inserted.
 41. The method of claim 28, wherein the AAVcapsid comprises the amino acid sequence of one of SEQ ID NOS:2-4. 42.The method of claim 29, wherein the AAV capsid comprises the amino acidsequence of one of SEQ ID NOS:2-4, wherein 10 or fewer amino acids aresubstituted, deleted, and/or inserted.
 43. The method of claim 29,wherein the AAV capsid comprises the amino acid sequence of one of SEQID NOS:2-4.
 44. The method of claim 34, wherein the AAV capsid comprisesthe amino acid sequence of one of SEQ ID NOS:2-4, wherein 10 or feweramino acids are substituted, deleted, and/or inserted.
 45. The method ofclaim 34, wherein the AAV capsid comprises the amino acid sequence ofone of SEQ ID NOS:2-4.
 46. The method of claim 35, wherein the AAVcapsid comprises the amino acid sequence of one of SEQ ID NOS:2-4,wherein 10 or fewer amino acids are substituted, deleted, and/orinserted.
 47. The method of claim 35, wherein the AAV capsid comprisesthe amino acid sequence of one of SEQ ID NOS:2-4.